Use of fructose-based therapies for the treatment of cancer

ABSTRACT

The methods and compositions of the invention are based on the preferential utilization of fructose by cancer cells. This invention relates to compositions, methods and kits utilizing fructose and other monosaccharides for the treatment of cancer. This invention also relates to methods and kits for using compositions to mimic or corrupt metabolic pathways of fructose and/or signal transduction pathways related to cancer cells for the treatment of cancer.

FIELD OF INVENTION

This invention relates to compositions, methods and kits utilizingfructose and other monosaccharides for the treatment of cancer. Thisinvention also relates to methods and kits for using compositions tomimic or corrupt metabolic pathways of fructose and/or signaltransduction pathways related to cancer cells for the treatment ofcancer.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

The prevalence of obesity in the United States, and worldwide isincreasing. Approximately 35 percent of U.S. adults 20 or older areoverweight (i.e., have a body mass index [BMI] of 25 to 29.9), and anadditional 30 percent are obese (i.e., have a BMI that is greater thanor equal to 30). Among children, an estimated 16 percent of childrenages 6-11 are overweight, over twice as many as two decades ago. Amongadolescents ages 12-19, the percentage of obesity has more than tripledfrom 5 to 16 percent. Although extreme obesity has received the mostattention in the clinical setting, moderate obesity is more common inthe general population. However, even moderate obesity can contribute tochronic metabolic abnormalities characteristic of the insulin resistancesyndrome, such as dyslipidemia, hypertension, insulin resistance, andglucose intolerance particularly when it is associated withintra-abdominal fat deposition (i.e., central obesity). Although it islikely that no single factor is responsible for the increased prevalenceof moderate obesity, it is likely that environmental elements areinteracting with predisposing genetic factors, and identification of theacquired causes contributing to an increase in the prevalence of obesityis key to developing public health policy and dietary and physicalactivity recommendations. Existing research indicates that weight,physical activity, and nutrition alter cancer risk and carcinogenesisfor many cancers, and evidence is accumulating on the effect of thesehealth factors on cancer prognosis and quality of life among cancersurvivors. A recent study of a very large prospective cohort of 900,000U.S. adults estimated that overweight and obesity in the US couldaccount for 14 percent of all deaths from cancer in men and 20 percentof those in women. An International Agency Research on Cancer (IARC)review entitled, “Weight Control and Physical Activity”, summarized theevidence across basic and population research, and estimated thatbetween one-quarter and one-third of the cases of many common cancersmay be attributable to the combined effect of increased body weight andinadequate physical activity.

The role of dietary changes as contributing factors to the developmentof obesity are under investigation, and along with an increase in totalenergy consumption over the past few decades, a clear shift in the typesof nutrients consumed in the American diet has been highlighted.Specifically, the consumption of fructose has increased dramatically,primarily because of increased consumption of beverages that are high infructose and the consumption of other foods such as breakfast cereals,baked goods, condiments, and prepared desserts sweetened with sucroseand high-fructose corn syrup (HFCS). HFCS is produced by the enzymaticisomerization of dextrose to fructose, and most HFCS used in beveragescontains about 55% fructose. Its commercial use increased in the 1970sso that by 1985, HFCS accounted for about 35% of the total amount ofsweeteners by dry weight in the food supply. Intakes based on the1977-1978 US Department of Agriculture Nationwide Food ConsumptionSurvey estimated the mean individual consumption of fructose inadolescents and adults was about 40 g/d, the range being 29-54 g/d.Thirteen of the 40 g of dietary fructose was estimated to come fromnaturally occurring sources of fructose, and 27 g from added sources offructose. More recent data on fructose consumption in the United Statesare not available, but food disappearance data, which can serve as anindicator of trends in consumption over time show that although the percapita use of sucrose has decreased moderately from 46.4 kg (102 lb) in1970 to 30.5 kg (67 lb) in 1997, the per capita use of HFCS hasincreased from a negligible 0.23 kg (0.5 lb) in 1970 to 28.4 kg (62.4lb) in 1997. This means that the combined consumption of sucrose, andhigh fructose corn syrup have increased by 26% from 64/g/day in 1970 to81/g/day in 1997. This represents an average daily energy intake fromadded fructose of about 1356 kJ (324 kcal). In fact, just two 355-mL(12-oz) soft drinks can supply up to 50 g/fructose (about 840 kJ, or 200kcal) or >10% of the daily energy requirements for an average-weightwoman, without considering any other dietary sources of fructose. Thus,fructose consumption now makes up a significant proportion of energyintake in the American diet, and this increase in fructose consumptionhas coincided with the increased prevalence of obesity over the past 2decades. These observations raise the question as to whether currentfructose intakes could contribute to weight gain and its metabolicsequelae, including cancer.

Pancreatic cancer is the fourth leading cause of death in the US (CzitoB. Willett C. Clark J. et al: Chemoradiation for unresectable pancreaticcancer. in Cameron J L (ed). Pancreatic Cancer. Hamilton. Ontario.Canada. B C Decker. 2001), 5-year survival is only 5%. Present molecularpathology, and cancer genetic studies indicate that pancreaticadenocarcinoma originates from pancreatic ductal cells, arises via aseries of progressive structural stages of neoplastic growth, termedpancreatic intraepithelial neoplasia (PanINs), that are precursors topancreatic adenocarcinomas, and associated with genetic alterationsoccurring in a temporal sequence (Bardeesy N, DePinho R A. PancreaticCancer Biology, and genetics Nat Rev Cancer 2: 897-909, 2002; Cubila AL, Fitzgerald P J. Morphological lesions associated with human primaryinvasive nonendocrine pancreas cancer. Cancer Res 36, 2690-9, 1976). Theearliest abnormalities include activating KRAS mutations, detectable in˜30% of the earliest PanIN (Kinstra D S, Longnecker D S. K-ras mutationsin pancreatic ductal proliferative lesions. Am J pathol 145, 1547-50,1994; Rozenblum E et al. Tumor-suppressive pathways in pancreaticcancer. Cancer Res 57, 1731-4, 1997), and altered epidermal growthfactor (EGF) signalling (both ERBB2 or Her2/neu, and ERBB3) (Korc M etal. Overexpression of the epidermal growth factor receptor in humanpancreatic cancer is associated with concomitant increases in the levelsof epidermal growth factor and transforming growth factor alpha. J ClinInvest 90, 1352-60 1992; Friess H et al. Pancreatic cancer: thepotential clinical relevance of growth factors and their receptors. JMol med 74; 35-42 1996; Siblia M et al. The EGF receptor provides anessential survival signal for SOS-dependent skin tumor development. Cell102, 211-220 2000). In late stage PaniNs, inactivation of INK4A(Rozenblum E et al. Tumor-suppressive pathways in pancreatic cancer.Cancer Res 57, 1731-4, 1997), and TP53 (Rozenblum E et al.Tumor-suppressive pathways in pancreatic cancer. Cancer Res 57, 1731-4,1997) are observed, the former cooperatively accentuating RASoncogenicity (Chin L et al. Cooperative effects of INK4A, and RAS inmelanoma susceptibility in vivo. Genes Dev 11, 2822-2834 1997), and thelatter facilitating genetic instability, including telomere dysfunction(Chin L et al. P53 deficiency rescues the adverse effects of telomereloss and coperates with telomere dysfunction to acceleratecarcinogenesis. Cell 97 527-538 1999). Inherited BRCA2 mutations,typically associated with familial breast, and ovarian tumors are foundin ˜17% of late stage pancreatic cancers in families harboring BRCA2mutations (Cancer risks in BRCA2 mutation carriers. The breast cancerlinkage consortium. J Natl Cancer Inst 91; 1310-1316 1999; Goggins M,Hruban R H, Kern S E. BRCA2 is inactivated in the development ofpancreatic intraepithelial neoplasia: evidence and implications. Am JPathol 156; 1767-1771, 2000.), and late PanINs frequently manifest lossof SMAD/DPC4, which encodes a key transcriptional regulator oftransforming growth factors-β family signaling (Hahn S A et al. DPC4, acandidate tumor suppressor gene at human chromosome 19q21.1. Science 271350-353 1996; Luttges J et al. Allelic loss is often the first hit inthe biallelic inactivation of the p53 and DPC4 genes during pancreaticcarcinogenesis. Am J Pathol 158 1677-1683 2001.).

The only well-established environmental etiologic factor is cigarettesmoking, although chronic pancreatitis has been reported to confer a20-fold excess risk (Lowenfels A B, Maisonneuve P, Cavallini G, et al.Pancreatitic and the risk of pancreatic cancer. N Engl J Med 1993; 328:1433-7), and evidence points to an association between diabetes mellitusand pancreas cancer, but whether these diseases are due to a commonexposure or are causally connected remains unknown (Anderson K E, PotterJ D, Mack T M. Pancreatic cancer. In: Schottenfeld D, Fraumeni J Jr,eds. Cancer epidemiology and prevention. New York, N.Y.: OxfordUniversity Press, 1996: 725-71; Everhart J, Wright D. Diabetes mellitusas a risk factor for pancreatic cancer: a meta-analysis. JAMA 1995; 273:1605-9). Additionally, higher fasting plasma glucose (>140 mg/dl) (Jee SH, Ohrr H, Sull J W, Yun J E, Samet J M. Fasting serum glucose level andcancer risk in Korean men and women. JAMA 2005; 293:194-202), orpostload (Gapstur S M, Gann P H, Lowe W, et al. Abnormal glucosemetabolism and pancreatic cancer mortality. JAMA 2000; 283: 2552-8)plasma glucose levels have been associated with increased pancreascancer mortality. A number of studies have investigated the role ofdietary factors in pancreatic cancer risk. As with other epithelialcancers, a diet high in vegetables and fruit- and perhaps specificallyhigh in folate has been associated with a lower risk, though notconsistently (World Cancer Research Fund Panel. Food, nutrition, and theprevention of cancer: a global perspective. Washington, D.C.: AmericanInstitute for Cancer Research, 1997; Nkondjock A, Krewski D, Johnson KC, Ghadirian P, and the Canadian Cancer Registries Epidemiology ResearchGroup. Dietary patterns and risk of pancreatic cancer. Int J Cancer May1; 114(5):817-823, 2005). Other studies have identified dietary intakeof lycopene or vitamin C as potentially protective factors (Nkondjock A,Ghadirian P, Johnson K C, Krewski D and the Canadian Cancer RegistriesEpidemiology Research Group. Dietary intake of lycopene is associatedwith reduced pancreatic cancer risk, J Nutr 135:592-597, 2004; Lin Y,Tamakoska A, Hayakawa T, Narus S, Kitagawa M and Ohno Y. Nutritionalfactors and risk of pancreatic cancer: A population-based case-controlstudy based on direct interview in Japan. J. Gastroenterol. Mar40(3):324-325, 2005). Some studies have reported increased pancreaticcancer risk associated with high cholesterol intake (Lin Y, Tamakoska A,Hayakawa T. Narus S, Kitagawa M and Ohno Y. Nutritional factors and riskof pancreatic cancer: A population-based case-control study based ondirect interview in Japan. J. Gastroenterol. Mar 40(3):324-325, 2005),and at least six case control studies have reported a positiveassociation between meat intake, and pancreatic cancer risk (Gapstur SM, Gann P H, Lowe W, et al. Abnormal glucose metabolism and pancreaticcancer mortality. JAMA 2000; 283: 2552-8; Howe G R and Burch J D.Nutrition and pancreatic cancer. Cancer Causes Control 7:69-82, 1996).However, in the large prospective 18-year follow-up Nurse Health Studyof 121,700 women, no relationship between total fat, fat type, andcholesterol was observed in the 178 women who developed pancreaticcancer (Michaud D S, Giovannucci E, Willett W C, Colditz G A and Fuchs CS. Dietary meat, dairy products, fat and cholesterol and pancreaticcancer risk in a prospective study. J Epidemiol 157(12):1115-1125,2003).

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with compositions and methods which are meantto be exemplary and illustrative, not limiting in scope.

The present invention provides for methods and kits of the treatment ofcancer.

One embodiment of the present invention provides for a method oftreating cancer in a mammal, comprising providing a composition capableof inhibiting or regulating a metabolic pathway of fructose; andadministering a therapeutically effective amount of the composition tothe mammal. In one embodiment, the composition may be a composition thatis capable of modulating an enzyme in the metabolic pathway of fructose.In another embodiment, the composition may be a composition that iscapable of modulating hexokinase, fructokinase-1, fructose-1-P adolase,transketolase or an analog thereof. In one embodiment, the compositioncapable of modulating transketolase is a small interfering RNA (siRNA)capable of suppressing transketolase mRNA expression.

In another embodiment, the composition may comprise a fructose analog,whereby the fructose analog competes with fructose to enter themetabolic pathway of fructose. In another embodiment, the compositionmay comprise a thiamine inhibitor. In another embodiment, thecomposition may comprise oxythiamine.

Another embodiment of the present invention provides for a method oftreating cancer in a mammal, comprising providing a composition capableof modulating a GLUT mRNA expression or a GLUT function; andadministering a therapeutically effective amount of the composition tothe mammal. In one embodiment, the GLUT may be GLUT-5. In anotherembodiment, the composition may comprise a small interfering RNA (siRNA)capable of suppressing the GLUT mRNA expression. In another embodiment,the composition is an antagonist of GLUT. In another embodiment, thecomposition is an antagonist of GLUT-5.

Another embodiment of the present invention provides for a method oftreating cancer in a mammal, comprising providing a fructose-based or afructose-analog based composition; and administering a therapeuticallyeffective amount of the fructose-based or the fructose-analog basedcomposition to the mammal, wherein the fructose-based or thefructose-analog based composition may comprise fructose conjugated to acompound. The compound may be a toxin, a cell signal deactivator, aradioactive agent or combinations thereof. In one embodiment, thedeactivator may be a cyclin-dependent kinase inhibitor. In variousembodiments, the compound may be conjugated to fructose at the first orthe second carbon atom. The toxin may be botulinum or diphtheria. Theradioactive agent may be any molecule or atom that emits radioactiverays; for example, gamma rays. Radioactive agents include but are notlimited to radioactive isotopes of carbon, oxygen, hydrogen, fluorine,iodine, gallium, technetium, indium and copper.

Another embodiment of the present invention provides for a method fortreating cancer in a mammal, comprising providing a compositioncomprising a radioactive isotope of fructose or a fructose analog; andadministering a therapeutically effective amount of the composition tothe mammal, wherein the radioactive isotope is incorporated into nucleicacid synthesis. In one embodiment, the radioactive isotope may becarbon-11 (¹¹C). In another embodiment the radioactive isotope may beoxygen 15 (¹⁵O).

Another embodiment of the present invention provides for a method fortreating cancer in mammal, comprising providing a fructose analogcapable of being cleaved into a toxic metabolite; and administering atherapeutically effective amount of the fructose analog to the mammal,whereby a cellular enzyme converts the fructose analog into the toxicmetabolite.

Other embodiments of the present invention provide for kits for thetreatment of cancer in a mammal. The kits may comprise an agent, whichmay be a composition capable of inhibiting or regulating a metabolicpathway of fructose; a composition capable of modulating a GLUT mRNAexpression or a GLUT function; a fructose-based or a fructose-analogbased composition, wherein the fructose-based or the fructose-analogbased composition may comprise fructose conjugated to a compound, whichmay be a toxin, a cell signal deactivator, a radioactive agent andcombinations thereof; a radioactive isotope of fructose or a fructoseanalog wherein the radioactive isotope is incorporated into nucleic acidsynthesis; or a fructose analog capable of being cleaved into a toxicmetabolite; and instructions to use the agent to treat cancer.

In one embodiment of the kit, the composition capable of inhibiting orregulating a metabolic pathway of fructose may be a composition capableof modulating hexokinase, fructokinase-1, fructose-1-P adolase,transketolase or an analog thereof. In another embodiment, thecomposition capable of inhibiting or regulating a metabolic pathway offructose may comprise a fructose analog, whereby the fructose analogcompetes with fructose to enter the metabolic pathway of fructose. Inanother embodiment, the composition capable of modulating the GLUT mRNAexpression may comprises a small interfering RNA (siRNA) capable ofsuppressing the GLUT mRNA expression. In another embodiment, thecomposition capable of modulating GLUT function may be an antagonist ofGLUT.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousfeatures of embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts a summary of potential mechanisms linking increasedrefined fructose consumption and cancer in accordance with an embodimentof the present invention.

FIG. 2 depicts a schematic of carbohydrate metabolism in accordance withan embodiment of the present invention. Glucose-derived carbon may enterthe glycolytic pathway in to generate pyruvate or lactate, a processlimited by feedback inhibition of citrate and ATP on the rate limitingenzyme phosphofructokinase. Fructose can also enter glycolysis viahexokinase to form fructose-6-phosphate or can also be metabolized byfructokinase, thereby allowing fructose derived carbon to enter theglycolytic pathway at the triose-phosphate level (left side of panel)(dihydroxyacetone phosphate and glyceraldheyde-3-phosphate). Thus,fructose may bypass the rate limiting phosphofructokinase control point,and can serve as an unregulated source of both glycerol-3-phosphate andacetyl CoA for hepatic lipogenesis.

FIG. 3 depicts the regulation of glycolysis and gluconeogenesis byfructose 2,6-bisphosphate (F2, 6BP) in accordance with an embodiment ofthe present invention. The major sites for regulation of glycolysis andgluconeogenesis are the phosphofructokinase-1 (PFK-1) andfructose-1,6-bisphosphatase (F-1,6-BPase) catalyzed reactions. PFK-2 isthe kinase activity and F-2,6-BPase is the phosphatase activity of thebi-functional regulatory enzyme,phosphofructokinase-2/fructose-2,6-bisphosphatase thatgenerates/dephosphorylates 2, 6 BP, the potent positive regulator ofglycolysis. Protein kinase A (PKA) is a cAMP-dependent protein kinasewhich phosphorylates PFK-2/F-2,6-BPase turning on the phosphataseactivity. (+ve) and (−ve) refer to positive and negative activities,respectively.

FIG. 4 shows that breast cancer cells proliferate more rapidly infructose (5 mM) than glucose (5 mM) containing medium in accordance withan embodiment of the present invention. Estrogen receptor (ER) positiveMCF-7, and ER negative MDA-MB231 cells were plated in 5 mM fructose orglucose for times between 8-72 h, and proliferative rates measured usingMTT assay. *, p<0.05; **, p<0.01.

FIG. 5 depicts fructose-mediated increased breast cancer proliferationin accordance with an embodiment of the present invention. ER positiveMCF-7, and ER negative MDA-MB231 cells were plated in a range offructose or glucose concentrations (5-25 mM) for 72 h, after whichproliferative rates were measured using an MTT assay. *, p<0.05 forconcentrations between 5-25 mM fructose versus glucose.

FIG. 6 depicts cell cycle profiles in accordance with an embodiment ofthe present invention. FACS analysis of MCF-7, and MDA-MB231 breastcancer cells after culture in 2 mM glucose, 25 mM glucose, and 25 mMfructose, depicting percentage of cells in G0/G1, and S-phase. *,p<0.01.

FIG. 7 depicts fructokinase-1 expression in accordance with anembodiment of the present invention. Breast cancer cells (MCF-7) expressFructokinase-1 (FK-1). RT-PCR was used to measure fructokinase-1 (FK-1)mRNA levels in MCF-7 cells following growth in 2 mM glucose, 5.5 mMglucose, or 5.5 mM fructose for 24 h, and in two human breast cancers(BT 1 & 2), and matched adjacent normal breast tissue (NB 1 & 2) fromthe same individual. Low level fructokinase was seen in human muscle.Human liver, and exclusion of template served as positive and negativecontrols.

FIG. 8 depicts fructose-1-P aldolase expression in accordance with anembodiment of the present invention. Breast cancer cells (MCF-7) expressthe β-isoform of fructose 1-P-aldolase (FPA). RT-PCR was used to measureFPA mRNA levels in MCF-7 cells following growth in 2 mM glucose, 5.5 mMglucose, or 5.5 mM fructose for 24 h, and in two human breast cancers(BT 1 & 2), and matched adjacent normal breast tissue (NB 1 & 2) fromthe same individual. As expected the FPA β-isoform was present in humanliver, and negative in human muscle tissue. Primer exclusion served asan additional negative control.

FIG. 9 depicts Northern blot analysis of fructokinase-1 mRNA levels inMCF-7 breast cancer cells in accordance with an embodiment of thepresent invention. 20 μg total RNA was used to measure FK-1 mRNA levelsin the MCF-7 cells following growth in 2 mM glucose, 5.5 mM glucose, or5.5 mM fructose for 24 h. FK-1 was also detected in human liver, withlower levels in muscle tissue.

FIG. 10 depicts GLUT-5 expression levels in accordance with anembodiment of the present invention. MCF-7 breast cancer cells wereincubated in 2 mM glucose (Glu), 5.5 mM glucose (Glu) or 5.5 mM fructose(Fru) for 4 or 24 hours, after which GLUT-5 expression was quantifiedusing real-time RT-PCR, normalized to GAPDH levels, and expressed asfold-increase compared to GLUT-5 levels detected in MCF-7 cells culturedin 2 mM glucose for a similar time period.

FIG. 11 depicts increased breast cancer phosphorylated-MEK,phosphorylated Rsk, and phosphorylated CREB in accordance with anembodiment of the present invention. MCF-7 breast cancer cells wereincubated in 2 mM glucose, 5 mM glucose or 5 mM fructose for 24 hours,after which Western blot analysis on total protein lysates was employedto examine components of the MAP kinase pathway. Increased breast cancerphosphorylated-MEK (pMEK), phosphorylated Rsk (pRsk), and phosphorylatedCREB (PCREB) levels were observed in MCF-7 cells after incubation in 5mM fructose compared to levels seen in 5 mM, and 2 mM glucose for 24 h.Total MEK, Rsk, and CREB levels, and β-actin served as loading controls.

FIG. 12 depicts an increase in AP1 activation in fructose-treated cellsin accordance with an embodiment of the present invention. MCF-7 breastcancer cells were incubated in 2 mM glucose, or 5 mM glucose or fructosefor 24 h, after which nuclear extracts were incubated with abiotinylated AP-1 probe in binding buffer. AP-1/AP1 probe complexes werenext conjugated to a streptavidin-coated assay plate and detected usingan AP-1 specific antibody, followed by binding of ahorseradish-peroxidase conjugated secondary antibody, and detection by acolorimetric reagent. *, p<0.001.

FIG. 13 depicts higher proliferation rates in pancreatic cancer cells infructose (5.5 mM) than glucose (5.5 mM) containing medium in accordancewith an embodiment of the present invention. PANC-1 cells werepre-incubated in 2 mM glucose, after which they were plated in 5.5 mMfructose or glucose for times between 12-72 h, after which proliferativerates were measured using an MTS assay. Proliferative rates areexpressed as the percentage difference between cells grown in 5.5 mMfructose versus 5.5 mM glucose. p<0.01 for times>48 h.

FIG. 14 depicts proliferation rates of pancreatic cells in differentfructose or glucose concentrations in accordance with an embodiment ofthe present invention. PANC-1 cells were plated in a range of fructoseor glucose concentrations (5-25 mM) for 72 h, after which proliferativerates were measured using an MTS assay. *, p<0.05 for concentrationsbetween 5-25 mM fructose versus glucose.

FIG. 15 depicts in the G0/G1 phase and the DNA synthesis (S) phase ofpancreatic cells in glucose and fructose in accordance with anembodiment of the present invention. PANC-1 cells were plated in 2, and5.5 mM glucose or 5.5 mM fructose for 48 h, after which cells were fixedin methanol, nuclei were stained with propidium iodide, and aliquotssubjected to FACS analyzes.

FIG. 16 depicts BRDU uptake following treatment with glucose or fructosein accordance with an embodiment of the present invention. PANC-1 cellswere pre-incubated in 2 mM glucose prior to treatment with 5.5 mMglucose or fructose for 72 h. BRDU (0.01 mg/ml) was added to the mediumat 48 h, and BRDU-uptake quantified at 72 h.

FIG. 17 depicts cell proliferation regardless of co-existent glucoseconcentration in accordance with an embodiment of the present invention.PANC-1 cells were pre-incubated in 2 mM glucose prior to treatment witha range of concentrations (0-15 mM) mixed with a range of fructoseconcentrations (0-15 mM) for 72 h. PANC-1 cell proliferative rates werethen measured using the MTS assay (absorbance in arbitrary units).

FIG. 18 depicts BRDU incorporation by pancreatic cells in accordancewith an embodiment of the present invention. Freshly resected pancreaticcancers were mechanically, and enzymatically dispersed, and cells werepre-incubated in 2 mM glucose prior to treatment with 5.5 mM glucose orfructose for 72 h. BRDU (10 μM) was added to the medium at 48 h, andBRDU-uptake quantified at 72 h.

FIG. 19 depicts PCNA expression in accordance with an embodiment of thepresent invention. PANC-1 cells were plated in 2 mM, and 5.5 mM glucose,or 5.5 mM fructose for 48 h, after which proliferating cell nuclearantigen (PCNA) levels were measured by Western blot analysis.

FIG. 20 depicts a degree of increased proliferation of normal pancreaticcells in accordance with an embodiment of the present invention. Freshlyresected normal pancreas tissues were mechanically and enzymaticallydispersed, and cells were pre-incubated in 2 mM glucose prior totreatment with 5.5 mM glucose or fructose for 72 h. BRDU (10 μM) wasadded to the medium at 60 h, and BRDU-uptake quantified at 72 h.

FIG. 21 depicts fructokinase-1 and fructose 1-P-adolase mRNA expressionin PANC-1 cells in accordance with an embodiment of the presentinvention. RT-PCR fructokinase-1 (FK-1), and fructose 1-P-aldolase (FPA)mRNA expression in PANC-1 cells, and a human pancreatic cancer following2 mM, or 5.5 mM glucose-, or 5.5 mM fructose-treatment.

FIG. 22 depicts GLUT-2 mRNA levels in pancreatic cancer cells inaccordance with an embodiment of the present invention. Quantitativereal-time RT-PCR was used to measure GLUT-5 mRNA levels in pancreaticcancer cells (PANC-1), following culture in 2 mM glucose, 5.5 mMglucose, or 5.5 mM fructose for 4 h and 24 h.

FIG. 23 depicts Western blot analysis examining MAPK pathway expressionin accordance with an embodiment of the present invention. PANC-1pancreatic cancer cells were incubated in 2 mM glucose, 5.5 mM glucoseor 5.5 mM fructose for 48 hours, after which Western blot analysis ontotal protein lysates was employed to examine phosphorylated MEK (pMEK),pRSK, and pCREB, and corresponding total MEK, RSK, and CREB levels.

FIG. 24 depicts luciferase activity of PANC-1 cells and primary cultureof surgically resected pancreatic tumor cells in accordance with anembodiment of the present invention. PANC-1 pancreatic cancer cells anda primary culture of a surgically resected pancreatic tumor wereincubated in 2 mM glucose, 5.5 mM glucose or 5.5 mM fructose for 48 h,after which nuclear extracts were incubated with a biotinylated AP-1probe, and detected by an antibody-linked colorimetric method. *, p<0.01

FIG. 25 depicts a schematic representation illustrating how carbohydratemass isoptomers may be utilized to map their metabolism via glycolysis,in this example to generate energy (ATP), pyruvate, which can enter thetricarboxylic acid cycle, and lactate, which is released into theculture medium in accordance with an embodiment of the presentinvention.

FIG. 26 depicts a schematic representation illustrating carbohydratemetabolism for lipid synthesis, and how carbohydrate mass isoptomers maybe utilized to map these pathways in accordance with an embodiment ofthe present invention.

FIG. 27 depicts a schematic representation of the steps via the pentosephosphate pathway, by which carbohydrates can be metabolized to providesugars for nucleic acid synthesis in accordance with an embodiment ofthe present invention.

FIG. 28 depicts the metabolism of fructose and glucose in pancreaticcancer cells in accordance with an embodiment of the present invention.Lactate generation from glycolysis (a), and hexose oxidation in thepentose and tricarboxylic acid cycles (b) following treatment of PANC-1cells with 2 mM glucose, 5.5 mM glucose, and 5.5 mM fructose for 72 h.In (a) the fraction of ¹³C-labeled lactate is expressed as thepercentage of total lactate measured demonstrating the contribution ofeach treatment to lactate generation. In (b), the ¹³C to ¹²C ratio inthe CO₂ released from the PANC-1 cells is presented.

FIG. 29 depicts fatty acid synthesis from glucose versus fructose inaccordance with an embodiment of the present invention. Fatty acidsynthesis represented by (a), ¹³C-labelled oleate, (b) ¹³C-labelledarachidic acid, and (c) ¹³C-labelled 24-carbon fatty acid fractionfollowing treatment of PANC-1 cells with 2 mM, and 5.5 mM glucose, and5.5 mM fructose for 72 h. For the fatty acids, the fraction of¹³C-labeled oleate, arachidic acid or 24-carbon fatty acid is expressedas percentage of total oleate, arachidic acid or 24-carbon fatty acids,respectively.

FIG. 30 depicts ribose and deoxyribose synthesis following glucose andfructose treatment in accordance with an embodiment of the presentinvention. Pentose phosphate shunt activity following treatment ofPANC-1 cells with 2 mM glucose, 5.5 mM glucose, and 5.5 mM fructose for72 h demonstrates that glucose was primarily metabolized via theoxidative pathway (regulated by glucose-6-phosphate dehydrogenase(G6PDH), which shuttles 5-carbon sugars back to the glycolytic pathway,whereas fructose was metabolized via the non-oxidative pathway, which isregulated by the enzyme transketolase (TKK), to synthesize nucleicacids.

FIG. 31 depicts substrate oxidation in accordance with an embodiment ofthe present invention. Substrate oxidation in a primary culture of anormal pancreas (left), and a pancreatic cancer (right) followingincubation in 2 mM, or 5 mM glucose, or 5 mM fructose for 72 h.

FIG. 32 depicts uric acid productionin PANC-1 cells in accordance withan embodiment of the present invention. Uric acid production inpancreatic cancer PANC-1 cells following incubation in 2 mM glucose, 5.5mM glucose, and 5.5 mM fructose for 24 h, 48, 72, and 96 h respectively,p<0.01 5.5 mM fructose versus 5.5 mM glucose.

FIG. 33 depicts Western blot analysis of transketolase expression inpancreatic cancer PANC-1 cells following incubation in 2 mM glucose(lanes 1 & 4), 5.5 mM glucose (lanes 2 & 5), and 5.5 mM fructose for 24h, and 48 h, respectively, in accordance with an embodiment of thepresent invention. Quantitation of TKK protein levels is depicted in thelower panel.

FIG. 34 depicts quantitation of transketolase activity in pancreaticcancer PANC-1 cells following incubation in 2 mM glucose, 5.5 mMglucose, and 5.5 mM fructose for 24 h, 48 h, and 72 h respectively, inaccordance with an embodiment of the present invention.

FIG. 35 depicts inhibition of pancreatic cancer PANC-1 cellproliferation by incubation with the transketolase inhibitor,oxythiamine (0.5-2 mM) for 72 h in accordance with an embodiment of thepresent invention. Proliferative rates were measured using the MTSproliferation assay.

FIG. 36 depicts random serum fructose, and glucose levels in 32 hospitalinpatients with unknown diagnosis in accordance with an embodiment ofthe present invention. Serum fructose was measured using an ELISA basedassay as described herein.

FIG. 37 depicts fasting serum fructose and glucose levels in patientswith pancreatic cancer and in normal volunteers in accordance with anembodiment of the present invention. (a) Fasting serum fructose andglucose levels in 6 patients with pancreatic cancer on the morning ofresection. (b) Serum fructose, and glucose levels (Mean±SEM) in 3 normalvolunteers after a 10 h fast, followed by ingestion of two cans of soda,containing 76 g of carbohydrate in the form of high fructose corn syrup(55% fructose, 45% glucose).

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); March, AdvancedOrganic Chemistry Reactions, Mechanisms and Structure 4th ed., J. Wiley& Sons (New York, N.Y. 1992); and Sambrook and Russel, MolecularCloning: A Laboratory Manual 3rd ed., Cold Spring Harbor LaboratoryPress (Cold Spring Harbor, N.Y. 2001), provide one skilled in the artwith a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

“Alleviating” specific cancers and/or their pathology includes degradinga tumor, for example, breaking down the structural integrity orconnective tissue of a tumor, such that the tumor size is reduced whencompared to the tumor size before treatment. “Alleviating” metastasis ofcancer includes reducing the rate at which the cancer spreads to otherorgans.

“Beneficial results” may include, but are in no way limited to,lessening or alleviating the severity of the disease condition,preventing the disease condition from worsening, curing the diseasecondition and prolonging a patient's life or life expectancy.

“Cancer” and “cancerous” refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. Examples of cancer include, but are not limited to, colorectalcancer, lung cancer, prostate cancer, hepatocellular cancer, gastriccancer, pancreatic cancer, cervical cancer, ovarian cancer, livercancer, bladder cancer, cancer of the urinary tract, thyroid cancer,renal cancer, carcinoma, melanoma, head and neck cancer, brain cancer,and breast cancer; including, but not limited to, ductal carcinoma,lobular carcinoma, Paget's disease, inflammatory breast cancer,medullary carcinoma, tubular carcinoma, mucinous carcinoma, cribriformcarcinoma, papillary carcinoma, and phyllodes tumors.

“Conditions” and “disease conditions,” as used herein may include, butare in no way limited to any form of cancer; in particular, pancreaticcancer and breast cancer, including but not limited to ductal carcinoma,lobular carcinoma, Paget's disease, inflammatory breast cancer,medullary carcinoma, tubular carcinoma, mucinous carcinoma, cribriformcarcinoma, papillary carcinoma, and phyllodes tumors.

“Curing” cancer includes degrading a tumor such that a tumor cannot bedetected after treatment. The tumor may be reduced in size or becomeundetectable, for example, by atrophying from lack of blood supply or bybeing attacked or degraded by one or more components administeredaccording to the invention.

“Fructose-based” as used herein includes compositions and therapieswhich include fructose or its analogs.

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be included within the scope of this term.

“Metabolic pathway” as used herein refers to a series of consecutiveenzymatic reactions that produce specific products.

“Pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

“Therapeutically effective amount” as used herein refers to that amountwhich is capable of achieving beneficial results in a patient withcancer. A therapeutically effective amount can be determined on anindividual basis and will be based, at least in part, on considerationof the physiological characteristics of the mammal, the type of deliverysystem or therapeutic technique used and the time of administrationrelative to the progression of the disease.

“Treatment” and “treating,” as used herein refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisease even if the treatment is ultimately unsuccessful. Those in needof treatment include those already with the Pathologic condition ordisease as well as those prone to have the pathologic condition ordisease or those in whom the pathologic condition or disease is to beprevented. In tumor (e.g., cancer) treatment, a therapeutic agent maydirectly decrease the pathology of tumor cells, or render the tumorcells more susceptible to treatment by other therapeutic agents, e.g.,radiation and/or chemotherapy.

“Tumor,” as used herein refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

The inventors' data show that fructose induces higher cancerproliferative rates than glucose, and their metabolomic studiesdemonstrate that cancer cells differentially utilize glucose andfructose, preferentially metabolizing fructose via transketolase fornucleic acid synthesis. The inventors believe that increased cancerproliferation rates due to fructose are not limited to pancreatic canceror breast cancer. Indeed, fructose induces high proliferation rates inall cancers since fructose may be utilized as an energy source. To placethese in vitro observations in the context of human disease, theinventors demonstrated that human circulating fructose concentrationsare at levels higher than those which mediate pro-proliferative effectsin pancreatic cancer, and that the capacity to control serum fructoselevels after a refined fructose load appears inferior to that whichcontrol blood glucose.

Embodiments of the present invention are based upon the inventors'discovery as described herein. The present invention includes methods,kits and models useful in the treatment and study of cancer in a mammal,such as a human, utilizing monosaccharide-based therapies and/orapproaches.

In various embodiments of the present invention, the monosaccharide is apentose and/or a hexose. Examples of pentose monosaccarides include butare not limited to, arabinose, lyxose, ribose, ribulose, xylose, andxylulose. Examples of hexose monosaccharides include but are not limitedto, allose, altrose, fructose, galactose, gulose, idose, mannose,psicose, sorbose, tagatose, and talose. In one embodiment, themonosaccharide may be fructose.

In other embodiments of the present invention, the monosaccharide may bea ketose. Examples of ketoses include but are not limited toerythrulose, ribulose, xylulose, fructose, psicose, sorbose, andtagatose. In one embodiment, the ketose is fructose.

In one embodiment of the present invention, the cancer may be breastcancer. In another embodiment of the present invention, the cancer maybe pancreatic cancer.

The fructose-based compositions and/or therapies may be administered byany appropriate technique, as will be readily appreciated by those ofskill in the art. By way of example and not to be interpreted aslimiting, the composition and/or therapy may be administered via oraladministration or intravenous administration.

Further embodiments include compositions, such as metabolites, and useof these compositions to mimic and/or corrupt metabolic pathways relatedto cancer cells.

Additional embodiments take advantage of the preferential use offructose for nucleic acid synthesis. Thus, in various embodiments, aradioactive isotope of fructose may be used to treat cancer, wherein theradioactive isotope is incorporated into nucleic acid synthesis. Due tothe instability of the isotope, the energy released by the isotope maydamage the DNA and may be toxic to the cell. The isotopic change may befor the carbon, the hydrogen, or the oxygen atom of fructose or fructoseanalog, or any combination of such atoms. One example is the C-11isotope (¹¹C). In another embodiment, ¹⁵O may be used. One of skill inthe art will appreciate appropriate isotopes that may be used withembodiments of the present invention.

Other embodiments of the present invention relate to the treatment ofcancer by identifying and administering compositions that inhibitvarious metabolic pathways related to monosaccharides and in particular,fructose. In one embodiment, a composition may be used to inhibit ametabolic pathway. Other embodiments include compositions and/orpeptides to block particular enzymes in metabolic pathways related tocancer cells. Various embodiments include compositions and/or peptidesto block hexokinase, fructokinase-1 (FK-1), fructose-1-P adolase (FPA)and transketolase. See FIG. 2.

Additional embodiments include analogs of fructose and use of theseanalogs to block fructose metabolism. The inventors believe that analogsof fructose may be used to compete with fructose and enter the metabolicpathway of fructose, and thus block fructose metabolism and thusdecrease an energy source for cancer cells, thereby reducing the growthof cancer cells. See FIG. 2.

Other embodiments of the present invention include monosaccharide-basedcompositions, use of monosaccharide-based compositions and methods fordelivery of monosaccharide-based compositions for the treatment ofcancer cells. Particular embodiments include fructose-basedcompositions. Further embodiments include linking a toxin (e.g., acytotoxin) or a radioactive agent to a monosaccharide, such as fructose,to target and kill cancer cells. Examples of toxins include but are notlimited to botulinum, and diptheria. Examples of radioactive agentsinclude but are not limited to carbon-11 (¹¹C) and iodine-131 (¹³¹I),fluorine, iodine, gallium, technetium, indium and copper. Otherembodiments include linking a compound to a monosaccharide, such asfructose, to target and inhibit the proliferation of cancer cells.

Another embodiment of the present invention provide for a method fortreating cancer in a mammal, comprising a fructose analog, wherein thefructose analog is capable of entering the cell and be converted into atoxic metabolite by an enzyme in the cell. In another embodiment, thefructose analog may be capable of entering the metabolic pathway offructose and may be converted into a toxic metabolite by an enzyme inthe metabolic pathway of fructose. For example, a fructose analog may bea fructose analog that is capable of being converted to a peroxidemolecule or is capable of being converted into a metabolite with aperoxide functional group, which may be toxic to the cells.

Other embodiments of the present invention are methods for delivery offructose-based therapeutic compositions for the treatment of cancercells by administering a therapeutically effective amount to a mammal.The inventors believe that fructose-based therapies, in which fructoseis preferentially used by the cancer cells is effective in targeting thecancer cells and thus provide beneficial results to the mammal.

Additional embodiments relate to the solute carriers such as glucosetransporters (GLUT). Particular embodiments relate to GLUT type 2(GLUT-2) and GLUT type 5 (GLUT-5), which facilitate cellular glucose andfructose uptake, respectively. Various embodiments include compositionsthat modulate GLUT mRNA expression. Other embodiments include methodsand approaches that modulate GLUT mRNA expression. In anotherembodiment, a siRNA approach is used to silence a GLUT that facilitatesfructose uptake. In one embodiment, a siRNA approach is used to silenceGLUT-5 expression. Further embodiments include compositions, methods andapproaches that regulate the amount of GLUT in the cells. Furtherembodiments include agonists and/or antagonists and use of agonistsand/or antagonists of GLUT to regulate the fructose uptake of cells. Theinventors have shown that cancer cells preferentially utilize fructoseas their energy source and have a higher proliferation rate in fructosethan in glucose. Thus, the inhibiting the uptake of fructose maydecrease the proliferation rate of cancer cells.

Further embodiments include fructose-based compositions containing cellsignal deactivators. These fructose-based compositions containingdeactivators may be preferentially utilized by cancer cells and thus thedeactivators may effect cell signaling. A cell signal deactivator maytarget cellular signals relating to the proliferation of the cell andmay be used to keep the cell in G1/G0 phase, rather than having the cellin the S1 phase. Deactivators such as cyclin-dependent kinaseinhibitors, p53 and p73 may be carried into the cancer cell usingfructose-based compounds to inhibit cell division. In anotherembodiment, as demonstrated by the inventors, transketolase is a keymediator of fructose-induced cancer cell proliferation. As such,therapies that target transketolase may be used to inhibit the growth ofcancerous cells. For example, silencing TKK RNA expression may inhibitthe growth of cancerous cells. In one embodiment, TKK may be silencedusing siRNA methods.

Another embodiment of the present invention provide for a method fortreating cancer in a mammal, comprising a fructose analog, wherein thefructose analog is capable of entering the metabolic pathway of fructoseand being converted into a toxic metabolite.

In various embodiments, the compositions utilized by the presentinventive methods may include a pharmaceutically acceptable excipient.“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients may be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, transmucosal, transdermal or parenteral.“Parenteral” refers to a route of administration that is generallyassociated with injection, including intraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection, or as lyophilized powders.

The pharmaceutical compositions according to the invention can alsocontain any pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier” as used herein refers to a pharmaceuticallyacceptable material, composition, or vehicle that is involved incarrying or transporting a compound of interest from one tissue, organ,or portion of the body to another tissue, organ, or portion of the body.For example, the carrier may be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it may come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The pharmaceutical compositions according to the invention can also beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulation, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous suspension. Such a liquid formulation may beadministered directly p.o. or filled into a soft gelatin capsule.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20th edition, Williams & Wilkins P A, USA) (2000).

Embodiments of the present invention are also directed to kits for thetreatment of cancer. The kit is an assemblage of materials orcomponents, including at least one of the compositions described hereinfor the treatment of cancer. Thus, in some embodiments the kit containsa composition including; for example, a composition capable ofinhibiting or regulating a metabolic pathway of fructose; a compositioncapable of modulating a GLUT mRNA expression or a GLUT function; afructose-based or a fructose-analog based composition, wherein thefructose-based or the fructose-analog based composition comprisesfructose conjugated to a compound selected from the group consisting ofa toxin, a cell signal deactivator, a radioactive agent and combinationsthereof; a radioactive isotope of fructose or a fructose analog whereinthe radioactive isotope is incorporated into nucleic acid synthesis; ora fructose analog capable of being cleaved into a toxic metabolite, asdescribed herein.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. In one embodiment, the kit isconfigured particularly for the purpose of treating mammalian subjects.In another embodiment, the kit is configured particularly for thepurpose of treating human subjects. In further embodiments, the kit isconfigured for veterinary applications, treating subjects such as, butnot limited to, farm animals, domestic animals, and laboratory animals.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to treat cancer. For example, instructions may includeinstructions to administer a therapeutically effective amount of acomposition, as described herein, to the mammal. Optionally, the kitalso contains other useful components, such as, diluents, buffers,pharmaceutically acceptable carriers, syringes, catheters, applicators,pipetting or measuring tools or other useful paraphernalia as will bereadily recognized by those of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging materials employed in the kit are those customarilyutilized in cancer treatment. As used herein, the term “package” refersto a suitable solid matrix or material such as glass, plastic, paper,foil, and the like, capable of holding the individual kit components.The packaging material generally has an external label which indicatesthe contents and/or purpose of the kit and/or its components.

Refined Carbohydrates, Fructose and Cancer Insulin Resistance andInsulin-Mediated Growth Promoting Actions

High carbohydrate intake has been hypothesized to be a risk factor forbreast cancer, possibly mediated by elevated levels of free insulin,estrogen and insulin-like growth factor-I. This hypothesis is supportedby population-based case-control studies. In a Mexican populationcharacterized by relatively high fat and high carbohydrate intakes,carbohydrate intake was positively associated with breast cancer risk,and the relative risk of breast cancer for women in the highest quartileof CHO-intake was 2.22 [95% confidence interval (95% Cl)1.63-3.04],compared with women in the lowest quartile, adjusting for total energyand potential confounding variables (P trend<0.0001) (Romieu I,Lazcano-Ponce E, Sanchez-Zamorano L M, Willett W, Hernandez-Avila M.Carbohydrates and the risk of breast cancer among Mexican women. CancerEpidemiol Biomarkers Prev 13:1283-9, 2004). This association was presentin both premenopausal and postmenopausal women, and among carbohydratecomponents, the strongest associations were observed for sucrose andfructose (Romieu I, Lazcano-Ponce E, Sanchez-Zamorano L M, Willett W,Hernandez-Avila M. Carbohydrates and the risk of breast cancer amongMexican women. Cancer Epidemiol Biomarkers Prev 13:1283-9, 2004).

In animal studies, chronic administration of a 60% fructose diet tonormal rats led to both hyperinsulinemia and in vivo insulin resistance.The fructose feeding-induced insulin resistance was mainly due to adiminished ability of insulin to suppress hepatic glucose output, andnot due to decreased insulin-stimulated glucose uptake by muscle (TobeyT A, Mondon C E, Zavaroni I, Reaven G M. Mechanism of insulin resistancein fructose-fed rats. Metabolism. 31:608-12, 1982). In other studiesfructose feeding to lean and obese Zucker rats, led to increased kidney,liver, and retroperitoneal adipose tissue weights, emphasizing thegrowth promoting effects of fructose (Koh E T, Mueller J, Osilesi 0,Knehans A, Reiser S Effects of fructose feeding on lipid parameter inlean, diabetic and nondiabetic Zucker rats. J. Nutr. 115: 1274-84,1985.).

In humans, epidemiological studies support an association betweenfructose-intake specifically, and cancer risk. Food-frequencyquestionnaires, documenting CHO intake, glycemic load, in addition tosucrose, and fructose intake, collected from a cohort of US women(n=88,802) participating in the Nurse' Health Study, revealed a 53%increased risk of pancreatic cancer development in women who had a highglycemic intake, and particularly in the cohort who reported a highfructose-intake (57% increased risk) (Michaud D S, Liu S, Giovannucci E,Willett W C, Colditz G A, Fuchs C. Dietary sugar, glycemic load, andpancreatic cancer risk in prospective study. J Natl Cancer Inst. 94:1293-300, 2002). This association was most pronounced in women whoadditionally had an increased BMI (>25) or sedentary lifestyle, andrelative risk in this group was 2.67 (95% Cl 1.02-6.99, p=0.03) forglycemic load, and 3.17 (95% Cl 1.13-8.91, p=0.04) for high fructoseintake, suggesting that high fructose intake may be an additive riskfactor on top of obesity (Michaud D S, Liu S, Giovannucci E, Willett WC, Colditz G A, Fuchs C. Dietary sugar, glycemic load, and pancreaticcancer risk in prospective study. J Natl Cancer Inst. 94: 1293-300,2002).

Fructose intake stimulates lipogenesis in animals, and although data inhumans is less conclusive, several studies have demonstrated significantincreases in lipid parameters on fructose versus glucose diets (Silveraet al., Glycemic index, glycemic load and pancreatic cancer risk(Canada). Cancer Causes Control May 16(4):431-436, 2005; Brand-Miller JC. Glycemic index in relation to coronary disease. Asia Pac J Clin Nutr13: S3, 2004; Laio et al., Genetic evidence for a common pathwaymediating oxidative stress, inflammatory gene induction, and aorticfatty streak formation in mice. J Clin Invest 94(2): 877-884, 1994;Vakkila et al., Inflammation and necrosis promote tumour growth. Nat RevImmunol. 4: 641-8, 2004; Calle et al., Overweight, obesity and cancer:epidemiological evidence and proposed mechanisms. Nat Rev Cancer 4:579-91, 2004).

The Fructose-Lipid Connection

The association between obesity and cancer, including but not limited tobreast and pancreatic cancer, is likely multifactorial, and in additionto insulin resistance, lipids and triglyceride levels have beenassociated with increased breast cancer risk. Several mechanisms bywhich elevated lipids, and triglycerides might increase breast cancerrisk have been proposed. For example, the hypertriglyceridemia, andaccompanying excess of circulating free fatty acids may contribute tothe development of a chronic low-grade inflammatory state, and recentstudies in cardiovascular disease have emphasized the role of markers ofsystemic inflammation as independent risk factors of coronary arterydisease. Similarly, more recent studies have begun to unravel the roleof low-level chronic systemic inflammation in the development, andprogression of solid tumors including breast cancer, and the NF-kappaBsignaling cascade is emerging as a key mediator in this regard (Laio F,Andalibi A, Qiao J H, Allayee H, Fogelman A M, and Lusis A J. Geneticevidence for a common pathway mediating oxidative stress, inflammatorygene induction, and aortic fatty streak formation in mice. J Clin Invest94(2): 877-884, 1994; Vakkila J, Lotze M T. Inflammation and necrosispromote tumour growth. Nat Rev Immunol. 4: 641-8, 2004). Thepro-inflammatory environment related to triglyceride and free fatty acidexcess promotes and supports tumorigenesis in multiple ways includingalteration in cell adhesion, platelet function, measures of oxidativestress, pro- and anti-inflammatory cytokines, and immune function (CalleE E, Kaaks R. Overweight, obesity and cancer: epidemiological evidenceand proposed mechanisms Nat Rev Cancer. 4: 579-91, 2004). Fructoseintake stimulates lipogenesis in animals, and although data in humans isless conclusive, several studies have demonstrated significant increasesin lipid parameters on fructose versus glucose diets (Sleder J, ChenY-ID, Cully M D, Reaven G M. Hyperinsulinemia in fructose-inducedhypertriglyceridemia in the rat. Metabolis 71:835-42, 1972; Zakim D. Theeffects of fructose on hepatic synthesis of fatty acids. Acta Med Scand542: 205-14, 1972; Bantle J P, Raatz S K, Thomas W and Georgopoulos A.Effects of dietary fructose on plasma lipids in healthy subjects 1-3. AmJ Clin Nutr 72 1128-1134, 2000; Henry R R, Crapo P A. Current issues infructose metabolism. Ann Rev Nutr 11: 21-39, 1991; Hollenbeck C B.Dietary fructose effects on lipoprotein metabolism and risk for coronaryartery disease. Am J Clin Nutr 58 (suppl) 800S-9S, 1993).

Carbohydrate Metabolism

Dietary carbohydrate from which humans gain energy enters the body incomplex forms, such as disaccharides and the polymers starch (amyloseand amylopectin) and glycogen. The first step in the metabolism ofdigestible carbohydrate is the conversion of the higher polymers tosimpler, soluble forms that can be transported across the intestinalwall and delivered to the tissues. The breakdown of polymeric sugarsbegins in the mouth by the enzyme lingual amylase. Once the food hasarrived in the stomach, acid hydrolysis contributes to its degradation.In the small intestine the main polymeric-carbohydrate digesting enzymeis pancreas-derived α-amylase, which has similar activity to salivaryamylase, producing disaccharides and trisaccharides. The latter areconverted to monosaccharides by intestinal saccharidases, includingmaltases that hydrolyze di- and trisaccharides, and the more specificdisaccharidases, sucrase, lactase, and trehalase. The net result is thealmost complete conversion of digestible carbohydrate to its constituentmonosaccharides. The resultant simple carbohydrates such as glucose, andfructose are transported across the intestinal wall to the hepaticportal vein and then to liver parenchymal cells and other tissues foroxidation in a process known as glycolysis.

Glucose and fructose are the two most important simple sugars for humanconsumption. They have the same molecular formula, C₆H₁₂O₆, but havedifferent structures. The sugars differ in the bond environment of theoxygen atom in the sugar, but each is a carbohydrate comprising 6 watermolecules and 6 carbon dioxide molecules with the yield of 6 oxygenmolecules. They are classified differently as hydrocarbon derivatives,glucose being classified as an aldehyde and fructose as a ketone, butare otherwise structurally identical. Although both glucose and fructosemetabolism is similar in many respects, there are important differencesthat the inventors believe may be important in cancer growth. Transportof glucose or fructose across the cell membrane is the firstrate-limiting step for sugar metabolism and is facilitated byglucose/fructose transport (GLUT) proteins, twelve (GLUT1-12) of whichhave been identified (Bantle et al., Effects of dietary fructose onplasma lipids in healthy subjects 1-3. Am J Clin Nutr 72: 1128-1134,2000). Once in the cell, glucose is oxidized to either lactate orpyruvate, and under aerobic conditions, the dominant product in mosttissues is pyruvate. In cancer cells, high glycolytic flux is requiredfor rapid tumor growth, and glucose uptake, and lactic acid levelscorrelate with tumor progression, invasiveness, patient morbidity, andmortality (Henry et al., Current issues in fructose metabolism. Ann RevNutr 11: 21-39, 1991; Hollenbeck C B. Dietary fructose effects onlipoprotein metabolism and risk for coronary artery disease. Am J ClinNutr 58 (suppl) 800S-9S, 1993; Tharanathan R N. Food-derivedcarbohydrates: structural complexity and functional diversity. Crit. RevBiotechnol. 22:65-84, 2002; Kim et al., Multifaceted roles of glycolyticenzymes Trends Biochem Sci. 30:142-50, 2005; Medina R A and Owen.Glucose transporters: Expression, regulation and cancer. Biol Res35(1):9-26, 2002; Gatenby R A, Gillies R J. Why do cancers have highaerobic glycolysis Nat Rev Cancer, 4(11):891-9, 2004.). Additionally,cancer cells maintain an abnormally high glycolytic rate even in thepresence of oxygen, a phenomena first described by Otto Warburg (Warburgeffect) (Eigenbrodt E. Glycolysis: one of the keys to cancer? TrendsPharmacol Sci 1: 240-245, 1980; Hennipman et al., Glycolytic enzymeactivities in breast cancer metastases. Tumour Biol 9: 241-248, 1988;Warburg et al., On the metabolism of cancer cells. Biochem Z 152:319-344, 1924; Bares et al., F-18 fluorodeoxygiucose PET in vivoevaluation of pancreatic glucose metabolism for detection of pancreaticcancer. Radiology, 192: 79-86, 1994). High glycolytic flux is requiredfor rapid tumor growth, and glucose uptake, and lactic acid levelsaccurately predict tumor progression, invasiveness, metastatic tendency,and overall patient morbidity, and mortality (Bares, R., Klever, P″HauPlrnann, S., Hellwig, D., Fass, J., Cremerius, U., Schurnpelick, V.,Mittemtayer, C., and Bull, U. F-18 fluorodeoxygiucose PET in vivoevaluation of pancreatic glucose Iretabolism for detection of pancreaticcancer. Radiology, 192: 79-86, 1994; Conti, P. S., Lilien, D. L.,Hawley. K., Keppler, J., Gmfton, S. T., and Bading, J. R. PET am[18F]-FDG in oncology: a clinical update. Nucl. Moo. Biol., 23: 717-735,1996; Walenta, S., Wetterling, M., Lehrlce, M., Schwicken, G., Sundfor,K., Rofstad, E. K., and Mueller-Klieser, W. High lactate levels in headand neck cancers predict likelihood of metastases, tumor recurrence, andrestricted patient survival in human cervical cancers. Cancer Res., 60:916-921, 2000; Walenta, S., Salameh, A., Lyng, H., Evensen, J. F.,MitZe, M., Rofslad, E. K., and Mueller-Klieser, W. Correlation of highlactate levels in head and neck tumors with incidence of metastasis. Am.J. Pathol., 150: 409-415, 1997; Di Chiro, G., DeLaPaz, R. L., Brooks, R.A., Sokoloff, L., Komblith, P. L., Smith, B. H., Patronas, N. J., Kufta,C. V., Kessler, R. M., Johnston, G. S. Manning, R. G., and Wolf, A. P.Glucose utilization of cerebral gliomas measured by [18F]fluoro-deoxyglucose and posilron emission tomography. Neurology, 32:1323-1329, 1982; Noguchi, Y., Saito, A., Miyagi, Y., Yamanaka, S.,Macat, D., Doi, C., Yoshikawa, T., Tsuburaya, A., Ito, T., and Satoh, S.Suppression of facilitative glucose transporter 1 mRNA can suppresstumor growth. Cancer Lett., 154: 175-182, 2000).

Fructose Metabolism

Diets containing large amounts of fructose or sucrose (a disaccharide ofglucose and fructose) can utilize the fructose as a major source ofenergy. Like glucose, fructose is taken into the cell by 3fructose-selective GLUTs 2, and 5, and 7. (Bantle et al., Effects ofdietary fructose on plasma lipids in healthy subjects 1-3. Am J ClinNutr 72: 1128-1134, 2000; Conti et al., PET am [18F]-FDG in oncology: aclinical update. Nucl. Moo. Biol., 23: 717-735, 1996; Walenta et al.,High lactate levels in head and neck cancers predict likelihood ofmetastases, tumor recurrence, and restricted patient survival in humancervical cancers. Cancer Res., 60: 916-921, 2000). Fructose can enterthe metabolic pathway in two ways, depending on which tissue is involved(muscle or liver). As most tissues contains only the enzyme hexokinase,fructose is phosphorylated at the sixth carbon atom, competing withglucose for the hexokinase involved (FIG. 2). However, fructose may alsobe phosphorylated on the first carbon atom in a reaction catalyzed bythe specific fructokinase-1, to generate fructose 1-phosphate (FIG. 2).Generally speaking, appreciable quantities of fructose are metabolizedby this route only in the intestines and the liver, but some studieshave postulated that cancer cells may possess fructokinase-1 activity(McGuinness O P, Chemington A D. Effects of fructose on hepatic glucosemetabolism. Curr Opin Clin Nutr Metab Care. 6: 441-8, 2003), and in thedata the inventors demonstrate that breast cancer cells expressfructokinase-1 mRNA. Fructose-1-phosphate can then be split intodihydroxyacetone phosphate and glyceraldehydes (FIG. 2). Theglyceraldehyde is next phosphorylated, and then along withdihydroxyacetone phosphate enters the latter steps of theEmbden-Meyerhof pathway. The significance of this is that fructose canenter the glycolytic pathway distal to the glycolysis rate limitingenzyme phosphofructokinase-1 to facilitate hepatic triacylglycerolproduction, and therefore unlike glucose, fructose can serve as arelatively unregulated source of acetyl-CoA. Alternatively,fructose-1-phosphate may also be phosphorylated to formfructose-1,6-diphosphate, and transit the glycolytic pathway in asimilar manner to glucose.

While not wishing to be bound by any particular theory, it is believedthat the glycolytic reactions (FIG. 2) catalyzed by hexokinase, andPFK-1 proceed with a relatively large free energy decrease. Although,these enzymes are allostearically controlled to regulate the fluxthrough glycolysis, the rate limiting step in glycolysis is the reactioncatalyzed by PFK-1 (McGuinness O P, Chemington A D. Effects of fructoseon hepatic glucose metabolism. Curr Opin Clin Nutr Metab Care. 6: 441-8,2003). PFK-1 is a tetrameric enzyme, and ATP is both a substrate and anallostearic inhibitor of PFK-1. Relatively recently, fructose2,6-bisphosphate, F2, 6BP, which is not an intermediate in glycolysis orin gluconeogenesis was identified as the most potent activator ofglycolysis (Kawaguchi T, Veech R L, Uyeda KRegulation of energymetabolism in macrophages during hypoxia. Roles of fructose2,6-bisphosphate and ribose 1,5-bisphosphate J Biol. Chem. 276:28554-61,2001; Hue L, Rousseau GGFructose 2,6-bisphosphate and the control ofglycolysis by growth factors, tumor promoters and oncogenesAdv EnzymeRegul. 33:97-110, 1993; Okar D A, Lange A J. Fructose-2,6-bisphosphateand control of carbohydrate metabolism in eukaryotes. Biofactors.10:1-14, 1999; Pilkis S J, Claus T H, Kurland I J, Lange A J.6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolicsignaling enzymeAnnu Rev Biochem. 64:799-835, 1995). This substrateexerts considerable control over the rate of glucose utilization byallostearically activating phosphofructo-1-kinase (PFK-1), andinhibiting the gluconeogenic enzyme fructose 1,6 bisphosphatase (F 1,6BPase-1) (FIG. 3). Synthesis, and breakdown of fructose 2,6 bisphosphate(Fru-2,6-BP) is catalyzed by the bifunctional enzyme6-phosphofructo-2-kinase/fructose 2,6 bisphosphatase (PFK-2/F 2,6 BPase)(FIG. 3). Four different genes coding different isoenzymes (PFKFB1-4)have been identified, and differ not only in their tissue distributionbut also in their kinetic, and regulatory properties (Manzano A, Perez JX, Nadal M, Estivill X, Lange A, and Bartrons R. Cloning, expression andchromosomal localization of a human testis6-phospho-fructo-2-kinase/fructose-2,6-biophosphatase. Gene (Amst.),229: 83-90, 1999; Heine-Suner D, Diaz-Guillen M A, Lange A G, andRodriguez de Corcoba S. Sequence and structure of the human6-phosphofructo-2-kinase/fructose-2,6-biophosphatase heart isoform gene(PFKFB2). Eur J Biochem, 254:103-110, 1998; Lange A G and Pilkis S G.Sequence of human liver 6-phosphofructo-2-kinase/fructose,2,6-biophosphatase. Nucleic Acids Res 18: 3652, 1990). Of the four PFK-2isoenzymes, only inducible PFK (iPFK/PFKFB-3) lacks a critical serinephosphorylation site that is required for the down-regulation of kinaseactivity (Sakakibara R, Kato M, Okamura N, Nakagawa T, Komada Y,Tominaga N, Shimojo M, and Fukusawa M. Characterization of a humanplacental fructose-6-phosphate, 2-kinase/fructose-2,6-biophosphatase. JBiochem (Tokyo) 122: 122-128, 1997). Consequently, PFKFB-3 has thehighest kinase:phosphatase activity ratio, and thus maintains highlyelevated F-2,6-BP levels, which in turn sustains high glycolytic rates.Significantly, iPFK/PFKFB-3 is constitutively expressed in several humancancer cell lines having high proliferative rates that require theactivity of the enzyme for the synthesis of 5-ribosyl-1-pyrophosphate, aprecursor for purine, and pyrimidines (Atsumi T, Chesney J, Metz C, LengL, Donnelly S, Makita Z, Mitchell R, Bucala R. High expression ofinducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-1;PFKFB3) in human cancers. Cancer Res 62: 5881-87, 2002; Chesney J,Mitchell R, Benigni F, bacher M, Spiegel L, Al-Abed Y, ham J H, metz C,Bucala R. An inducible gene product for 6-phosphofructo-2-kinase with anAU-rich instability element: role in tumor cell glycolysis and theWarburg effect. Proc Natl Acad Sci, USA 96: 3047-3052, 1999). Indeed,the activity of this enzyme has been proposed as part of the explanationfor the Warburg effect, where high glycolytic rates are observed intransformed cells even in the presence of oxygen (Darville M I. Crepin KM, Vandekerckhove J, Van-Damme J, Octave I N, Rider M H, Marchand M J,Hue L, and Rousseau G G. Complete nucleotide sequence coding for ratliver 6-phosphofructo-2-kinase/fructose-2,6-bis phosphatase derived froma cDNA clone. FEBS Lett 224: 317-321, 1987). As noted, rapidlyproliferating transformed cells constitutively express iPFK-2 (PFKFB-3)mRNA and protein in vitro, and inhibition of iPFK-2 expression decreasestumor growth in experimental animal models (Perez J X, Roig T, ManzanoA, Dalmau M, Boada J, Ventura F, Rosa J L, Bermudez J, Bartrons R.Overexpression of fructose 2, 6 bisphosphatase decreases glycolysis anddelays cell cycle progression. Am J Physiol Cell Physiol 279: C1359-65,2000). In addition, iPFK-2 (PFKFB-3) is induced by hypoxia, and theiPFK-2 (PFKFB-3) promoter has response elements to several transcriptionfactors including myc, and NfkappaB (Norris J L and Baldwin A S Jr.Oncogenic ras enhances NF-κB transcriptional activity throughRaf-dependent and Raf-independent mitogen-activated protein kinasesignaling pathways. J Biol Chem 274: 13841-13846, 1999; Minchenko A,Leschinsky I, Opentanova I, Sang N, Srinivas V, Armstead V and Caro J.Hypoxia-inducible factor-1-mediated expression of the6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) gene.Its possible role in the Warburg effect. J Biol Chem 277: 6183-6187,2002).

In T47-D breast cancer cells, progestin treatment led to increased PFK-2expression, and increased the number of cells in S-phase (Hamilton J A,Callaghan M J, Sutherland R L, and Watts C K. Identification of PRG1, anovel progestin-responsive gene with sequence homology to6-phospho-fructo-2-kinase/fructose, 2,6-biophosphatase. Mol Endocrinol11: 490-502, 1997). In summary, fructose conversion to fructose 2,6bisphosphate by the isoforms of PFK-2 acts to speed transit of fructose,and glucose through the glycolytic pathway to increase energyavailability for cell proliferation. While not wishing to be bound byany particular theory, the inventors believe that the increasedfructose-induced breast cancer proliferative rates are due to increasedinducible PFK-2 (iPFK-2/PFKFB-3) levels generatingfructose-2,6-bisphophate that in turn potently stimulatesphosphofructo-1-kinase, thereby transiting both glucose and fructosefaster through the glycolytic pathway to generate increased energy forcell proliferation. The data demonstrate that MCF-7 breast cancer cellsexpress fructokinase-1 (FK-1). This enzyme potentially enables breastcancer cells to metabolize fructose directly to fructose-1-phosphate(FIG. 2), to enter the glycolytic pathway distal to the glycolysis ratelimiting enzyme phosphofructokinase-1, and to generate higher ATP levelsfor cell proliferation. Therefore, unlike glucose, fructose can serve asa relatively unregulated source of acetyl-CoA.

Additionally, the data demonstrate pancreatic cancer FK-1, andfructose-1-P-aldolase mRNA expression, and increased GLUT-5 mRNA levelsfollowing fructose-treatment.

While not wishing to be bound to any particular theory, it is believedthat the form in which fructose is ingested, is extremely important.Clearly, fruit, and vegetables contain fructose, but this is mixed withsignificant quantities of fiber, which limits fructose absorption rates,and contains antioxidant vitamins, particularly vitamins C and A, andadditional components that provide anticancer benefits. While notwishing to be bound by any particular theory, the inventors believe thatit is dietary sources of refined fructose that are the real risk, in theform of fructose corn syrup. The inventors also believed that theincreased proliferative actions of fructose are not restricted to breastcancers with disrupted EGFR-signaling.

While not wishing to be bound by any particular theory, the inventorsalso believed that refined fructose is an independent risk factor for invivo breast cancer development and progression. The effects of fructosein vivo may be multifactorial and include effects due to increasedinsulin resistance, effects due to an enhanced inflammatory state, andeffects due to associated obesity.

In vitro data demonstrated that breast cancer cells proliferate morerapidly in medium containing fructose than glucose, and expressfructo-kinase-1 (FK-1), a glycolytic enzyme that is usually only foundin significant amounts in normal hepatic and intestinal cells. While notwishing to be bound by any particular theory, it is believed thataberrant breast cancer FK-1 expression and increased expression of asecond key glycolytic enzyme, inducible phospho-fructokinase-2(iPFK-2/PFKFB-3) underlie the increased fructose-mediated proliferationthe inventors have observed. Fructo-kinase-1 expression allows breastcancer cells to metabolize fructose to fructose-1-phosphate, therebybypassing key rate limiting steps that regulate glucose metabolism. Thesecond enzyme, iPFK-2/PFKFB-3 generates fructose 2, 6 bisphosphate (F2,6 BP), which is a potent inducer of phosphofructokinase-1 activity,the rate-limiting enzyme of glycolysis.

The inventors' demonstration (FIG. 10) that fructose may be able toself-regulate its own transporter, GLUT-5, support the rationale to useSiRNA approaches to silence GLUT-5 expression or transiently overexpress GLUT-5 in the breast cancer cells, and examine proliferativerates in fructose-treated breast cancer cells. The inventors havedemonstrated that breast cancer cells express significant levels offructose kinase-1 (FK-1), and fructose-1-P aldolase (FPA), two enzymesthat potentially enable cancer cells to metabolize fructose byadditional glycolytic pathways to glucose, and emphasizing theimportance of the proposed study of the metabolic pathways utilized byfructose, and glucose respectively. Further, Western blot analysis usingphospho-specific antibodies to pMEK, p90RSK, and pCREB levels in proteinlysates harvested from MCF-7, and MDA-MB231 cells cultured in fructose,and glucose for 24 h indicate that the MAPK pathway is involved infructose-mediated increased breast cancer proliferative rates (FIG. 11),and support the examination of transcriptional activation in fructose-,and glucose-treated MCF-7, and MDA-MB231 cells, transiently transfectedwith CRE-luciferase, AP-1-luciferase, and SRE-luciferase reporterconstructs.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Breast Cancer Cells Exhibit Increased In Vitro ProliferativeRates in Fructose Versus Glucose

Standard media for breast cancer cell culture contain either 5-7 mM(low) and 12-25 mM (high) glucose concentrations. Estrogen receptor (ER)positive MCF-7, and ER negative MDA-MB231 cells were cultured overnightin 2 mM glucose standard DMEM medium containing 10% fetal bovine serumprior to incubation for periods between 8, and 72 hours in standard DMEMmedium containing 10% fetal bovine serum, and 5 mM glucose or 5 mMfructose.

Proliferative rates were then measured in replicate (minimum of 6)aliquot wells of the breast cancer cells grown in glucose, or fructosecontaining medium. As depicted in FIG. 4, both ER negative MDA-MB231,and ER positive MCF-7 breast cancer cells exhibited higher proliferativerates in 5 mM fructose, compared to cells grown in 5 mM glucose, butotherwise identical conditions. In separate experiments, proliferativerates of the MCF-7, and MDA-MB231 breast cancer cells were comparedfollowing culture in a range of fructose, and glucose concentrations(2-25 mM) for 72 hours. As depicted in FIG. 5, the fructose-mediatedincreased breast cancer proliferation was observed over a range ofconcentrations of fructose, and as depicted in FIG. 5, cells grown for72 hours in 5 mM fructose exhibited higher proliferative rates to cellsgrown in 5-fold higher glucose concentrations (25 mM).

Example 2 Breast Cancer Cells Express Fructokinase-1 mRNA

While not wishing to be bound by any particular theory, it is believedthat the increased breast cancer proliferative rates are due toincreased expression of several key glycolytic enzymes, includingfructokinase-1, and inducible phosphofructokinase (iPFK-2/PFKFB-3)levels. The inventors used RT-PCR analysis and specific primer pairs(SEQ ID NO: 1 (3′cctgccagatgtgtctgcta), SEQ ID NO: 2(5′aagtgcttggccacatcttt)) to examine fructokinase-1 (FK-1) mRNA levelsin glucose- or fructose-treated breast cancer cells. As depicted in FIG.7, MCF-7 cells cultured in 2 mM glucose exhibited minimal FK-1 mRNAexpression, slightly higher levels were seen in cells grown in 5 mMglucose, but highest FK-1 levels were seen in breast cancer cells grownin 5 mM fructose. As expected human hepatic tissue exhibited high, andmuscle low FK-1 mRNA expression. Also examined were FK-1 mRNA levels intwo surgically resected human breast cancers, and paired adjacent normalbreast tissue from the same individual. FK-1 mRNA was detectable in bothnormal and tumor tissue.

Example 3 Breast Cancer Cells Express Fructokinase-1 and Fructose-1-PAldolase mRNA

FK-1 catalyzes the conversion of fructose to fructose-1-phosphate, thefirst step by which fructose can enter the fatty acid synthesis pathway.The data demonstrate that breast cancer cells also express fructose1-P-aldolase (FPA) mRNA, the second enzyme in this pathway. As depictedin FIG. 8, RT-PCR analysis employing specific primer pairs for theβ-isoform of FPA demonstrated FPA mRNA expression using total RNAderived from MCF-7 cells cultured in 2 mM glucose, 5.5 mM glucose, and5.5 mM fructose. As expected human hepatic tissue exhibited FPA mRNA,whereas muscle, which does not express the FPA-isoform, was negative.Also observed was FPA mRNA expression in two surgically resected humanbreast cancers and in paired adjacent normal breast tissue from the sameindividual. These results support the inventors' belief that breastcancer cells can potentially utilize fructose via alternative metabolicpathways to glucose to generate higher ATP levels for the enhancedgrowth rates.

In additional studies, the inventors used Northern blot to confirmsignificant FK-1 mRNA expression in the breast cancer cells. As depictedin FIG. 9, fructokinase-1 mRNA expression was detected in MCF-7 cellscultured in 2, and 5.5 mM glucose, and 5.5 mM fructose, and in humanmuscle and liver tissue. Highest FK-1 expression was observed in livertissue and intriguingly in the MCF-7 cells cultured in 5.5 mM fructose,suggesting that fructose treatment may actually induce this keymetabolic enzyme for fructose metabolism via the alternative pathwaydescribed herein.

Example 4 Fructose Treatment Induces GLUT-5

Cellular fructose uptake is facilitated by a member of solute carrierfamily 2, called glucose transporter type 5 (GLUT-5). Followingincubation of MCF-7 breast cancer cells in 2 mM, 5.5 mM glucose or 5.5mM fructose for 48 h, total RNA was harvested, and analyzed byquantitative real-time RT-PCR analysis using GLUT-5 specific primers toquantify GLUT-5 mRNA expression. GLUT-5 expression was normalized toGAPGH mRNA expression, and expressed as fold-change in comparison tocontrol breast cancer cells incubated in 2 mM glucose for 4, and 24hours. As depicted in FIG. 10, no significant change in GLUT-5 mRNAexpression was observed following incubation of the breast cancer cellsin 5.5 mM glucose or 5 mM fructose for 4 hours, but after 24 hourincubation, a two-fold increase in the specific fructose transporter,GLUT-5, was observed in cells incubated in 5.5 mM fructose compared toGLUT-5 expression in cells incubated in 5.5 mM glucose.

Example 5 Fructose-Treatment Induces the MAPK Pathway in Breast CancerCells

Although several signal transduction pathways are likely involved in thefructose-mediated enhanced breast cancer growth and while not wishing tobe bound by any particular theory, the inventors believe that theMAP-kinase pathway is involved. The inventors have used Western blotanalysis to examine the MAPK pathway in breast cancer cells incubated in2 mM glucose, 5.5 mM glucose, and 5.5 mM fructose for 48 hours. MEKmediated phosphorylation of the p44/42 MAP kinase (ERK) is the firststep in this cascade and the inventors observed increased expression ofphospho-MEK (pMEK) in MCF-7, and MDA-MB231 breast cancer cells grown in5.5 mM fructose, in comparison to cells grown in 5.5, and 2 mM glucose.Activation of MEK, and subsequently ERK results in phosphorylation ofp90^(Rsk), which leads to transcriptional activation of CREB and AP1,which bind to cAMP or AP1 response elements respectively, activatinggene transcription and promoting cellular growth. After 24 h incubationof breast cancer cells in 5.5 mM fructose, the inventors observedincreased phospho-Rsk (pRsk), and phospho-CREB (pCREB), in comparison tocells grown in 5.5, and 2 mM glucose for the same time period.Furthermore, using an ELISA-based transbinding AP1 assay (Panomics), theinventors demonstrated a 3-fold increase in AP1 activation in 5.5 mMfructose-treated cells in comparison to a 2.2 fold increase in cellstreated with 5.5 mM glucose (FIG. 12; *p<0.001). These results providefurther insight into the mechanism(s) for the fructose-induced increasedbreast cancer proliferation that were seen.

Example 6 Characterization of the Mechanism(s) of Fructose-EnhancedBreast Cancer Proliferation In Vitro

Firstly, ER-positive MCF-7, and ER-negative MDA-MB231 breast cancercells are cultured in a range of concentrations of glucose, and fructoseto confirm, and extend the findings that culture in fructose results inincreased breast cancer growth. The data (FIG. 5) shows that breastcancer cells exhibit higher proliferative rates at all fructoseconcentrations between 2.5, and 25 mM.

Briefly, MCF-7, and MDA-MB231 cells (˜10,000 per well) are first platedon to 96-well plates, in standard whole serum (10% FBS), high glucose(25 mM) DMEM medium. Cells are then incubated overnight in whole serum(10% FBS) low glucose (2 mM) DMEM. The following day, cells are changedto medium containing a range of glucose or fructose (5, 10, 25, 50, 75,& 100 mM) for times 24, 48, 72 and 96 h. Proliferative rates aremeasured in the cells using the MTS assay. Briefly, cells are incubatedwith ‘One Solution’ (containing tetrazolium compound MTS and phenazineethosulfate reagent) at 37° C. for 2 h. Absorbance (490 nm) whichcorrelates closely with cell proliferation measured using CellTiter 96AQueous One Solution Cell Proliferation Assay (MTS) according tomanufacturer's instructions (Promega, Madison, Wis., USA). This rapidassay is ideal for these large numbers of samples, and quickly allowsdetermination of optimal dose ranges, and treatment times for subsequentexperiments. The focus is on physiologically relevant glucose & fructoseconcentrations (5-25 mM), to translate these findings to human breastcancer. Proliferative rates are also quantified by measurement ofbromodeoxyuridine (BRDU) uptake in the fructose-, and glucose-treatedcells. Breast cancer cells are also plated in 12-well dishes, incubatedin a similar range of glucose, and fructose concentrations for 24-96 h,and aliquots of the fructose-, and glucose-treated cells then preparedfor FACS analysis to measure cell-cycle profiles.

Example 7 Fructose/Glucose Uptake and Oxidative Phosphorylation

Two areas are important: (1) Increased GLUT expression resulting inincreased fructose uptake into the cell; and (2) Increased expression ofkey glycolytic enzymes, particularly fructokinase-1, and induciblephosphofructokinase-2, that result in increased oxidativephosphorylation.

GLUT Expression

As noted above, a family of glucose-uptake and transporter (GLUT)proteins regulate cellular glucose uptake, and previous studies havedemonstrated increased GLUT expression in cancer, including thefructose-selective GLUT-5 (Zamora-Leon S P, Dolde D W, Concha I I, RivasC I, Delgado-Lopez F, Baselga J, Nualart F, Carlos-Vera J. Expression ofthe fructose transporter GLUT5 in human breast cancer. Proc Natl AcadSci 93: 1847-52, 1996; Macheda M L, Rogers S, and Best J D. Molecularand cellular regulation of glucose transporter (GLUT) proteins incancer. J Cellular Physiol 202: 654-662, 2005; Smith T A D. Facilitativeglucose transporter expression in human cancer tissue. Brit J Biomed Sci56(4): 285-292, 1999; Rogers S, Docherty S E, Slavin J L, Henderson andBest J D. Differential expression of GLUT12 in breast cancer and normalbreast tissue. Cancer Letters 193(2): 225-233, 2003). IncreasedGLUT-mediated fructose uptake, thereby increasing substrateavailability, contributes to increased proliferation. Therefore, breastcancer GLUT expression is examined RT-PCR and Western blot analysis inthe fructose- and glucose-treated breast cancer cells. While not wishingto be bound by any particular theory, it is believed that the increasedfructose-mediated proliferation is due to enhanced GLUT-5-traffickedfructose uptake, and fructose is able to self-regulate its owntransporter. SiRNA approaches are employed to silence GLUT-5 expressionin the breast cancer cells and to determine the alteration in increasedgrowth rates observed in the fructose-treated breast cancer cells. Inparallel experiments, vectors harboring GLUT-5 are transientlytransfected into MCF-7, and MDA-MB231 cells. Increased GLUT-5 expressionare confirmed by Western blot, and the effects of increased GLUT-5expression on breast cancer cell growth rates across a range of fructoseand glucose concentrations examined.

Oxidative Phosphorylation

Three approaches are used to examine oxidative phosphorylation in thefructose-treated and glucose-treated breast cancer cells. Firstly,several key glycolytic enzymes following breast cancer fructose-, orglucose-treatment, is quantitated using RT-PCR, and Northern blotanalysis. While not wishing to be bound by any particular theory, it isbelieved that there is increased glycolysis, and hence oxidativephosphorylation in the fructose-versus glucose-treated cells, one wouldexpect that expression of the key glycolytic enzymephospho-fructokinase-1 is increased in the fructose-treated breastcancer cells, compared to the glucose-treated cells. Briefly, afterglucose- or fructose-treatment, total RNA (1-2 μg) from the MCF-7, andMDA-MB231 breast cancer cells are treated with DNAse I, at 37° C. for 30min and reverse transcribed using SuperScript First-Strand Synthesissystem for RT-PCR (Invitrogen). Specific oligonucleotide primer pairsare used to amplify phospho-fructokinase-1 expression in the glucose- orfructose-treated cells (PFK-1: SEQ ID NO: 3 (3′agcctccctatccaggaaaa) andSEQ ID NO: 4 (5′tagacagcagccaggacctt)) using polymerase chain reaction(PCR). PCR for 18S is performed as an internal control on both RTpositive and negative samples to confirm cDNA product integrity.Aliquots of the PCR products are electrophoresed on 1% agarose gels andstained with ethidium bromide to visualize PCR products. Band intensityis quantified using scanning densitometry.

While not wishing to be bound by any particular theory, it is believedthat the increased glycolysis is due to increased cancer fructosekinase-1 (FK-1), and inducible phosphofructokinase-2 (iFPK-2) activity.Northern blot analysis is used to quantify FK-1, and iPFK-2/FBPase-2.Briefly, total RNA is extracted from the fructose-, or glucose-treatedMCF-7, and MDA-MB231 breast cancer cells in Trizol, and Northern blotanalyses using full-length FK-1, or iPFK-2/FBPase-2 [α-32P]dCTP—randomprimer labeled cDNA as probes, performed by standard procedures (HeaneyA P, Singson R, McCabe C J, Nelson V, Nakashima M, Melmed S. PituitaryTumor Transforming Gene: a novel marker in colorectal tumors. Lancet355:716-719, 2000). RNA integrity is verified by observing the rRNAbands in ethidium bromide gels under UV, and the level of mRNA isquantified by densitometric scanning of the autoradiograms and correctedfor 18 s rRNA expression (Sambrook J, Fristsch E F, and Maniatis T.Molecular Cloning: A Laboratory Manual, edited by Nolan C. Cold SpringHarbor, N.Y.: Cold Spring Harbor, p. 7.3-7.5, 1989).

If inducible phosphofructokinase-2 (iFPK-2) activity is increased,increased levels of the metabolite fructose-2, 6 bisphosphate(Fru-2,6-P2) in the fructose—compared to glucose- or vehicle-treatedcells may be observed. Therefore, Fru-2,6-P2 levels are quantitated.Briefly, fructose-, or glucose-treated MCF-7, and MDA-MB231 cells arehomogenized in 0.1 M NaOH, heated to 80° C. for 15 min, and centrifugedat 12,000 g for 5 min. Fru-2,6-P2 is then determined in the supernatantsby its ability to activate pyrophosphate-dependent iPFK-1 from potatotubers as described by Van Schaftingen et al. (Van Schaftingen E.Fructose 2,6-bisphosphate. Adv Enzymol Relat Areas Mol Biol 59: 315-395,1987; Van Schaftingen E, Lederer B, Bartrons R, and Hers H G. A kineticstudy of pyrophosphate: fructose-6-phosphate phosphotransferase frompotato tubers. Application to a microassay of fructose 2,6-bisphosphate.Eur J Biochem 129: 191-195, 1982; Van Schaftingen E, Jett M F, Hue L,and Hers H G. Control of liver 6-phos-phofructokinase by fructose2,6-biosphophate and other effectors. Proc Natl Acad Sci USA 78:3483-3486, 1981). The glycolytic metabolites are then measuredspectrophotometrically in neutralized perchloric extracts, usingstandard enzymatic methods.

As discussed above, fructose can potentially bypass the rate-limitingstep (catalyzed by PFK-1) of glycolysis, if it is metabolized directlyto fructose-1-phosphate by the enzyme fructokinase-1. The inventors'data demonstrates that breast cancer MCF-7 cells express FK-1 mRNA.Therefore, Northern blot is used to quantify fructokinase-1, andfructose 1-P aldolase mRNA levels, the second enzyme in this pathway.The latter catalyzes the conversion of fructose-1-phosphate todihydroxyacetone phosphate, and its presence confirms that breast cancercells can traffic fructose directly into the lipogenesis pathway,bypassing regulatory controls that restrict glycolytic glucosetrafficking. To determine the activity of fructokinase-1 (FK-1),fructose 1-phosphate levels, the metabolite of the FK-1 catalyzedreaction, are measured Fructose 1-phosphate is measured using acommercially available assay based on its conversion to fructose1,6-bisphosphate by a bacterial fructose-1-phosphate kinase. Briefly,the open reading frame encoding Escherichia coli Fru1PK has beenintroduced in an expression plasmid (pET3a) based on the T7promoter-driven system, and is used to overexpress the enzyme, and thepreparation can be used in an enzymatic assay to measure specificallyfructose 1-phosphate levels in cell or tissue extracts.

Thirdly, lactate production, as this is a key metabolite of glycolysis,and as noted previously serum lactate levels correlate with severaloutcome measures in human cancer, is measured. For measurement oflactate production, cell suspensions attached to microcarriers are used.Briefly, after incubation of the breast cancer cells in fructose orglucose, cells are trypsinized, washed, and added to preswollenCytodex-1 microcarriers (Pharmacia Biotech), and suspended in DMEM. Toachieve a maximum yield of cells attached to microcarriers, the culturesare stirred for 5 min every 30 min. After 2 h, the medium is changed andthe cultures stirred continuously at 60 rpm. Microcarrier cultures arethen incubated for up to 48 h. Cells attached to microcarriers are thenrinsed and suspended in Krebs bicarbonate buffer containing 2.5 mMCaC12, 2% BSA, and 10 mM glucose prior to measurement of lactateproduction.

Expression vectors harboring full-length wild-type cDNA fromiPFK-2/FBPase-2, and a truncated mutant iPFK-2/FBPase-2 that onlyexhibits bisphosphatase activity (pFBPase-2) are generated. Wt- andMut-iPFK-2/FBPase-2 are transiently transfected into MCF-7, andMDA-MM231 cells, and expression is confirmed by RT-PCR analysis usingspecific sense and antisense Wt-, and Mut-primers (Darville M I. CrepinK M, Vandekerckhove J, Van-Damme J, Octave I N, Rider M H, Marchand M J,Hue L, and Rousseau G G. Complete nucleotide sequence coding for ratliver 6-phosphofructo-2-kinase/fructose-2,6-bis phosphatase derived froma cDNA clone. FEBS Lett 224: 317-321, 1987). Wt-, and Mut-MCF-7, andMDA-MB231 transfectants are incubated in fructose, or glucose for 24 h,and effects of overexpression of the Wt-, or Mut-iPFK-2/FBPase-2 onproliferative rates are examined. As Wt-iPFK-2/FBPase-2 furtherincreases Fru-2,6-P2 levels to further induce PFK-1, and while notwishing to be bound by any particular theory, it is believed that breastcancer cells transfected with the Wt-iPFK-2/FBPase-2 will exhibit higherproliferative rates than vector-transfected cells, and the proliferativerates will be further increased by fructose-treatment. While not wishingto be bound by any particular theory, the inventors believe thatMut-iPFK-2/FBPase-2 expression drives glycolysis towards glycogensynthesis, reduces proliferative rates, and abrogates thefructose-induced breast cancer proliferative rates.

Example 8 Signal Transduction

Although it is likely that several signal transduction pathways areinvolved in fructose-mediated breast cancer proliferation, previousstudies have demonstrated that ras-transformation in rat-1 fibroblastsled to increased F2, 6 BP levels, and aerobic glycolysis, and multiplepotential binding sites for several ras-activated transcription factors,including myc, and nuclear factor kB have been identified on the iPFK-2promoter (Norris J L and Baldwin A S Jr. Oncogenic ras enhances NF-κBtranscriptional activity through Raf-dependent and Raf-independentmitogen-activated protein kinase signaling pathways. J Biol Chem 274:13841-13846, 1999; Minchenko A, Leschinsky I, Opentanova I, Sang N,Srinivas V, Armstead V and Caro J. Hypoxia-inducible factor-1-mediatedexpression of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3(PFKFB3) gene. Its possible role in the Warburg effect. J Biol Chem 277:6183-6187, 2002). Therefore, while not wishing to be bound by anyparticular theory, the inventors believe that the MAP-kinase pathway islikely to be important. Several MAPK cascades have been identifiedincluding the extracellular signal-related kinase pathways (ERK 1/2,ERK5), and the stress activated kinase pathways (JNK/SAPK, p38 MAPK). Asall MAPK pathways operate through sequential phosphorylation events,Western blot analysis using phospho-specific antibodies is employed toexamine p90^(RSK), CREB, c-fos, and ELK-3 levels in protein lysatesharvested from MCF-7, and MDA-MB231 cells cultured in 2, 5, 10, and 25mM fructose, and glucose for 24 h. Additionally, MCF-7, and MDA-MB231cells are transiently transfected with CRE-luciferase, AP-1-luciferase,and SRE-luciferase reporter constructs. A β-Galactosidase reporterconstruct is co-transfected as a control. Following incubation of thetransient transfectants in a range of glucose, and fructoseconcentrations as before (5, 10, & 25 mM) for 24 h, cell lysates areharvested, and luciferase activities measured, and normalized for μ-Galto correct for changes in cell number or transfection efficiency. Allsamples are analyzed in triplicate, a minimum of three separateexperiments are conducted for each study outlined above, andstatistically analyzed as described below. To further characterize therole of the MAP-kinase pathway in fructose-mediated breast cancerproliferation, proliferative rates in the fructose-, and glucose-treatedbreast cancer cells with, and without pharmacological inhibition of theMAPK-pathway using the specific MKK 1/2 and, MEK 1/2 inhibitors PD98059,and UO126, are compared. Additionally, plasmids encoding a dominantnegative MAPK, or empty vector alone (Pearson G, Robinson F, Gibson T B,Xu B, Karandikar M, Berman K, Cobb M H. 2001. Mitogen-activated protein(MAP) kinase pathways: Regulation and physiological functions. Endo Rev22: 153-183) are transiently co-transfected into MCF-7 and MDA-MB231breast cancer cells before incubation in fructose or glucose. Dominantnegative (DN) MAPK expression is confirmed by western blot, andproliferative rates in fructose-treated DN MAP-kinase expressing breastcancer cells compared with glucose-treated DN MAP-kinase expressingcells, and fructose-, and glucose-treated control vector transfectedcells. Protein aliquots from fructose-, and glucose-treated breastcancer cells are also analyzed to examine activation of other signaltransduction pathways, including the PI-3K/AKT and phospholipase-C-γpathways, as these pathways are extremely important in breast cancer(Mills G B, Kohn E, Lu Y, Eder A, Fang X, Wang H, Bast R C, Gray J,Jaffe R, Hortobagyi G Linking molecular diagnostics to moleculartherapeutics: targeting the PI3K pathway in breast cancer Semin Oncol.30 (Suppl 16): 93-104, 2003).

Example 9 Determine Effects of Refined Fructose Consumption on In VivoBreast Cancer Growth

The effects of 10% and 20% added refined fructose and glucose on breastcancer development are examined. These percentages are chosen as theyare conservative but meaningful equivalent to current human refinedcarbohydrate intake. Therefore, refined glucose or fructose isadministered to mice overexpressing activated neu oncogene under thecontrol of the mouse mammary tumor virus (MMTV) promoter. Effects oftreatments on time to tumor development and tumor multiplicity areexamined.

Example 10 Transgenic Mice

Female MMTV-erbB2 transgenic mice are purchased from the JacksonLaboratory. In this murine breast cancer model, the MMTV promoter fromthe mouse mammary tumor virus long terminal repeat causes the erbB2 geneto be expressed in the mammary gland. These animals reproducibly developfocal mammary tumors that arise spontaneously at 4 months, with a medianincidence of 205 days. Mice are housed in animal facilities, inaccordance with institutional care and use guidelines.

Example 11 Treatment and Data Collection

The mice receives a commercially purchased standard chow diet(Labdiet.com, certified rodent diet 5002), eat ˜5 g per day, and receivea total daily intake of approximately 1700 Kcal, comprising 64.5% CHO,11.8% fat, 23.5% protein, and 4.6% fiber. Mice also receive a fructoseor glucose sugar solution containing 0.42 g or 0.84 g fructose orglucose (diluted in 100 ml) which equate to 10% or 20% added refinedcarbohydrate respectively or vehicle daily from age 3 months to 12months. Developing tumors in the fructose-, glucose-, and vehicle-fedmice are measured twice a week with electronic calipers, and tumorvolumes determined by multiplying the square of the width (w) by thelength (l) and dividing by two (i.e., (w²l)/2). Individual animalweights, tumor size and tumor location are recorded for each animaltwice a week. Animals are euthanitized when they develop tumors of 1000mm3 or more or at the end of the experiment, at the same time of day (9am) to limit temporal variations in glucose, insulin, and otherparameters. Two hours before killing, the mice are injectedintraperitoneally with bromodeoxyuridine (3 mg/mL) in phosphate-bufferedsaline, (100 μL/10 g body weight). The end of the experiment is definedas the time when all vehicle-treated mice have developed a tumor(usually at ˜330 days of age). At that time, all remaining mice(vehicle-, glucose-, and fructose-treated) are killed, tumors resected,and studied as below.

Example 12 Histology and Biomarker Analysis—Breast Cancer

For histology, breast tumors are fixed in 10% formalin and embedded inparaffin. Tissue sections are mounted on slides and processed forhematoxylin-eosin staining. Immunohistochemical staining is performedfor erbB2 to confirm tumor expression, and for proliferating cellnuclear antigen (PCNA & Ki-67) to compare tumor proliferative rates inthe mice. Briefly, tissue sections are deparaffinized in xylene,rehydrated, endogenous peroxidase activity blocked, and non-specificbinding reduced with goat serum. Sections are then incubated with one ofthe following antibodies: rabbit anti-c-erbB2 antibody (Neomarkers)(1:50), or monoclonal anti-PCNA Or Ki-67 (MIB-1) antibodies (1:200).Washed sections are incubated with biotinylated goat anti-rabbitantibody, incubated with the ABC kit (Vector labs), and3-amino-9-ethylcarbazole to visualize the peroxidase complex. Levels ofpRb are evaluated by visual assessment with a semiquantitative scoringsystem rating staining intensity (from 0 to 3). Staining forbromodeoxyuridine (BRDU) is performed with the DAKO Animal Research Kitsystem. Briefly, tissue sections are prepared, endogenous peroxidaseblocked, and nonspecific binding reduced as before. BRDU are stainedwith a mouse anti-BRDU monoclonal antibody (DAKO). Slides are incubatedwith streptavidin-horseradish peroxidase, and visualized withdiaminobenzidine chromagen. Counterstaining is performed withhematoxylin. Stained sections are reviewed, and scored by counting thepositive and negative cells in 10 high-powered fields in tissue samplesfrom 4 mice from each group. Results are expressed as an averagepercentage with 95% confidence intervals.

Example 13 Glucose, Insulin, IGF1, Lipid, and Inflammatory CytokineLevels

Fructose-, glucose-, and vehicle-treated mice are fasted for 24 h (9 am-9 am) every 4 weeks, and at euthanasia, blood are collected by the tailclip method into chilled tubes for assay of glucose, insulin, lipid andinflammatory cytokine levels. Plasma glucose concentrations aredetermined using Glucotrend 2 (Roche Diagnostics); plasma insulin (LincoRes), and IGF-1 Phoenix Pharmaceuticals levels are measured using solidphase two site enzyme immunoassays; plasma triglyceride, free fattyacids, lactate, and μ-hydroxybutyrate concentrations are analyzed usingcommercially available kits (Sigma Diagnostics). Serum inflammatorymarkers include C-reactive protein, IL-6 (R&D Systems), serum sialicacid, soluble intracellular and vascular adhesion molecules (sICAM-1,and sVCAM-1) (Bender MedSystems Inc), and amyloid A levels, and areassayed using commercially purchased ELISA based kits (R & D,Quantikine). All parameters are measured in triplicate in the breastcancer susceptible mice, and means compared between the fructose-, andglucose-, and vehicle-fed animals, at the various timepoints using ANOVAwith Bon-Ferroni post t-tests for multiple comparisons. Time curves aregraphed to compare trends in fasting glucose, insulin, lipid, andtriglyceride levels, and inflammatory markers over the course of dietarytreatment.

Example 14 Alternative Approaches

Allotypic graft tumors are generated by injecting cells derived fromerbB2 Tg mice (NK), into the mammary gland of FVB/N mice (these are thebackground strain).

Example 15 Pancreatic Cancer Cells Exhibit Increased In VitroProliferation in Fructose in Comparison to Glucose

Standard media for maintenance cancer cell line culture, includingpancreatic cancer cells such as PANC-1 cells contain either low—(5-7mM), or high—(12-25 mM) glucose concentrations. Clearly, apart from thesetting of hyperglycemia, as in patients with diabetes, the glucoseconcentrations in which pancreatic cancer cells are maintained in vitroare much higher than normal physiological conditions, which rangebetween 2-5 mM glucose. While the inventors did not wish to expose thecells to serum-free conditions, as is customary when testing potential“growth-factor” effects, the inventors sought to conduct the experimentsunder conditions that might ultimately be physiologically relevant topatients with pancreatic cancer. Therefore, the inventors firstpre-treated the pancreatic cancer cells overnight in standard DMEMmedium containing low physiological glucose concentrations (2 mMglucose), and standard 10% fetal bovine serum. The pancreatic cancercells were then incubated for times between 12, and 96 hours in standardDMEM medium containing 10% fetal bovine serum, and 5.5 mM glucose or 5.5mM fructose. Proliferative rates were then measured in replicate(minimum of 6) aliquot wells of the pancreatic cancer cells, and thechange (expressed as percent increase) in proliferative rates comparedbetween cells grown in glucose, or fructose containing medium. Asdepicted in FIG. 13, PANC-1 pancreatic cancer cells began to exhibithigher proliferative rates in 5.5 mM fructose, compared to cells grownin 5.5 mM glucose by 24 h, the difference in proliferative ratescontinued to increase, reaching ˜13% at 48 h, was maximal at 72 h, bywhich time pancreatic cancer cells demonstrated a 23% higherproliferative rate in 5.5 mM fructose than 5.5 mM glucose, but inotherwise identical conditions (p<0.01 for all times>48 h).

This first result intrigued the inventors, but given there is littledocumentation regarding physiological fructose concentrations, theinventors first considered that the “apparent” pro-proliferative effectsof fructose were simply a consequence of the highly stylized in vitroexperimental conditions, and that if the pancreatic cancer cells werecultured in higher glucose concentrations, this would result in similarproliferative rates to those the inventors observed following culture in5.5 mM fructose. Therefore, in the next experiments, the inventorscompared proliferative rates of the PANC-1 pancreatic cancer cellsfollowing culture for 72 h in a range of fructose, and glucoseconcentrations (2-25 mM). This timepoint was chosen as the inventors hadseen maximal pancreatic cancer cell proliferation in 5.5 mM fructose at72 h. As depicted in FIG. 14, higher glucose concentrations (up to 25 mMglucose) still did not result in the same magnitude of increase inpancreatic cancer cell proliferation as that which was induced byfructose concentrations from 5 mM upwards. In fact, cells grown for 72hours in 5 mM fructose exhibited higher proliferative rates than cellsgrown for 72 h in 5-fold higher glucose concentrations (25 mM) (FIG.14). The fructose-mediated increased pancreatic cancer proliferation wasfirst demonstrable around 5 mM fructose, and interestingly not appearingto increase further with higher fructose levels. While not wishing to bebound by any particular theory, the inventors believe that this latterfinding may be because the PANC-1 cells are proliferating at close tomaximal capacity by 72 h, once they are exposed to comparatively lowfructose concentrations (5 mM). MTS assay was employed to measurepancreatic cancer cell proliferative rates, which utilizes mitochondrialoxidation to quantify cell proliferation. As the inventors wereexamining carbohydrate-mediated effects, the inventors considered thechange observed in proliferative rates, as measured by this assay,simply reflected changes in mitochondrial oxidation. Therefore, twoadditional methods were utilized to examine proliferative ratesfollowing fructose-treatment. Firstly, the inventors examined cell cycleprofiles in the pancreatic cancer cells following culture in 5.5 mMglucose, or 5.5 mM fructose for 48 h, after which cells were fixed inmethanol, and stained with propidium iodide prior to flow-activatedcytometric analysis (FACS) to determine the percentage of cells in theresting G0/G1 phase, and the DNA synthesis (S) phase. If the sugars wereinducing pancreatic cancer cell proliferation, the inventors wouldexpect to see an increased percentage of cells in the S-phase, with aconcordant reduced percentage of cells in the G0/G1 phase. As depictedin FIG. 15, following culture in 5.5 mM glucose, an increase of ˜8% ofcells in S-phase was observed, in comparison to PANC-1 cells cultured in2 mM glucose, in keeping with some increase in cell proliferation.However, following incubation of the PANC-1 cells in 5.5 mM fructose, amore striking increase (˜17%) in the percentage of PANC-1 cells in theproliferative S-phase (PANC-1:2 mM glucose, 30.9%; 5.5 mM glucose,38.5%; 5.5 mM fructose, 47%) was observed. In conjunction with increasedS-phase population, a concordant reduction in cells in the resting G0/G1(PANC-1:2 mM glucose, 34.5%; 5.5 mM glucose, 26.2%; 5.5 mM fructose,21.2%) was noted (FIG. 15), supporting the MTS assay data, andindicating that the pancreatic cancer cells exhibited higherproliferative rates when cultured in equivalent fructose and glucoseconcentrations.

As a further measure of PANC-1 proliferative rates, the inventorscompared bromodeoxyuridine (BRDU) uptake following PANC-1 cell treatmentas before with 2 mM, and 5.5 mM glucose, or 5.5 mM fructose for 72 h. Inthe final 12 h, 10 μM BRDU was added to the glucose-, andfructose-treated cells, after which BRDU-positive cells were counted. Asdepicted in FIG. 16, in agreement with the other measures of pancreaticcancer cell proliferation, some increase in BRDU uptake was noted in the5.5 mM glucose-treated pancreatic cancer cells in comparison to 2 mMglucose-treated cells. However, significantly higher BRDU-uptake (˜75%higher than with 5.5 mM glucose) was noted in the PANC-1 cells which hadbeen treated with 5.5 mM fructose in comparison to cells treated with5.5 mM glucose, confirming that fructose leads to higher pancreaticcancer cell proliferation than equivalent glucose concentrations. Afurther question the inventors sought to address was would thefructose-induced pancreatic cancer proliferative rates be maintained ifthe PANC-1 cells were also exposed to, and able to utilize theircustomary culture glucose concentrations. Therefore, the inventorscultured PANC-1 cells for 72 h in a range of glucose concentrations(0-15 mM) admixed with a range of fructose concentrations (0-15 mM).

As depicted in FIG. 17, once the PANC-1 cells were exposed to fructoseconcentrations of 6 mM and above, an increase in cell proliferation wasnoted, regardless of the co-existent glucose concentration. Thissuggested to the inventors that the pancreatic cancer cells were in someway able to distinguish glucose and fructose, and utilize the lattersugar in a different manner to lead to higher proliferative rates. Basedon this series of experiments, the inventors concluded that fructoseinduced greater in vitro pancreatic cancer proliferative rates incomparison to equivalent, and even significantly higher glucoseconcentrations. As immortalized cell-lines, such as PANC-1 pancreaticcancer cells, can become dedifferentiated after repeated passages, andin order to translate these findings in the PANC-1 cells to patientswith pancreatic cancer, the inventors also examined proliferative ratesin primary cultures of fresh surgically resected pancreatic cancers,following incubation in 2 mM, and 5.5 mM glucose or 5.5 mM fructose.Briefly, after mechanical and enzymatic (trypsin and DNAse)disaggregation, aliquots of tumor cells were seeded in 6-well plates,and incubated in standard DMEM media with 10% fetal bovine serum, and 2mM glucose, 5.5 mM glucose, or 5.5 mM fructose for 72 h. As before inthe PANC-1 experiments, BRDU (10 μM) was added in the final 12 h ofincubation in the sugars, following which BRDU-uptake was quantified.All 5 primary pancreatic cancers have exhibited higher proliferativerates in 5.5 mM fructose compared to 5.5 mM glucose, as illustrated in arepresentative tumor in FIG. 18.

Additionally, protein extracts were harvested in RIPA buffer from thePANC-1 cells following 2 mM, 5.5 mM glucose-, and fructose-treatment for48 h, analyzed by Western blot, and immunoblotted using a specificmonoclonal antibody to proliferating cell nuclear antigen (PCNA). Asdepicted in FIG. 19, some increased PCNA expression was observed in thePANC-1 cells following incubation in 5.5 mM glucose, compared to 2 mMglucose, in keeping with increased cell proliferation as the inventorshad demonstrated with BRDU-uptake, and MTS assays. However, highest PCNAlevels were observed in protein extracts derived from PANC-1 cellstreated with 5.5 mM fructose, compared to 5.5 or 2 mM glucose,supporting the FACS and MTS proliferation assay findings, and confirminghigher pancreatic cancer proliferative rates in equimolar fructoseversus glucose.

Example 16 Normal Pancreatic Cells do not Exhibit IncreasedProliferative Rates Following Fructose-Treatment

In the course of obtaining fresh surgically resected pancreatic cancertissue, the inventors also obtained some peripherally adjacent “normal”pancreatic tissue and normal tissue from a patient undergoing pancreasresection for complications of chronic pancreatitis. The inventorsstudied 5 of these “normal” pancreas tissues, and although caveats mayexist given that they were derived from pancreases which were involvedwith another disease entity, the inventors believe they offer a usefulcomparator for the tumors. Similar to the pancreatic cancers, normalpancreatic tissue was mechanically and enzymatically dispersed, andcells seeded into 6-well plates containing 2 mM glucose, 5.5 mM glucose,or 5.5 mM fructose for 72 h. As before, BRDU (10 □M) was added, afterwhich BRDU-uptake was quantified. In parallel experiments, whensufficient normal pancreatic issue was available, proliferative ratesfollowing 72 h incubation in the sugars were also measured using the MTSproliferation assay. As depicted in FIGS. 20A and B, derived from twodistinct representative normal pancreas tissues, the normal pancreascells did demonstrate some increase in proliferation (measured by bothBRDU-uptake (FIG. 20A) or MTS assay (FIG. 20B)) following incubation in5.5 mM glucose, and 5.5 mM fructose, in comparison to proliferativerates seen in 2 mM glucose. However, contrary to what the inventors hadobserved in the PANC-1 cells and primary pancreatic cancer cells,proliferative rates did not differ significantly between normal pancreascells incubated in 5.5 mM glucose or 5.5 mM fructose. These studies innormal pancreatic cells raise the intriguing possibility that thedifferential proliferative response to fructose versus glucose isrestricted to transformed pancreatic cancer cells, and has importantimplications for therapeutic applications described herein.

Example 17 Pancreatic Cancer Cells Express Fructokinase-1, andFructose-1-P Aldolase mRNA

Firstly, the inventors believed that pancreatic cancer cells may expressfructokinase-1 and fructose-1-P aldolase, enzymes that would enable themto metabolize fructose to fructose-1-phosphate, allowing fructose toenter the TCA cycle downstream of the negative regulatory feedback ofphosphofructokinase. To examine this, the inventors used RT-PCR analysisand specific primer pairs to examine fructokinase-1 (FK-1) andfructose-1-P aldolase (FPA) mRNA levels in glucose- or fructose-treatedPANC-1 cells, and in surgically resected human pancreatic cancers. Asdepicted in FIG. 21, both PANC-1 cells, and the primary pancreaticcancers (representative tumor in lane 4) cultured in 2 mM, and 5.5 mMglucose, and 5.5 mM fructose did express FK-1, and FPA mRNA, enzymesthat would potentially enable them to metabolize fructose todihydroxyacetone phosphate, thereby bypassing key rate limiting steps towhich glucose is subjected, to generate higher ATP levels forproliferation.

Example 18 Fructose-Treatment Increase Pancreatic Cancer GLUT-5Expression

A family of facilitative glucose/fructose transporter (GLUT) proteinsregulate cellular sugar uptake, and previous studies have demonstratedincreased cancer GLUT expression, including the fructose-selectiveGLUT-5 (Zamora-Leon S P, Dolde D W, Concha I I, Rivas C I, Delgado-LopezF, Baselga J, Nualart F, Carlos-Vera J. Expression of the fructosetransporter GLUT5 in human breast cancer. Proc Natl Acad Sci 93:1847-52, 1996; Macheda M L, Rogers S, and Best J D. Molecular andcellular regulation of glucose transporter (GLUT) proteins in cancer. JCellular Physiol 202: 654-662, 2005; Smith T A D. Facilitative glucosetransporter expression in human cancer tissue. Brit J Biomed Sci 56(4):285-292, 1999; Rogers S, Docherty S E, Slavin J L, Henderson and Best JD. Differential expression of GLUT12 in breast cancer and normal breasttissue. Cancer Letters 193(2): 225-233, 2003). Therefore, the inventors'believed that if pancreatic cancer cells exhibited increased GLUTexpression, this may increase carbohydrate (fructose) substrateavailability, to mediate increased pancreatic cancer proliferation.Teal-time RT-PCR was used to measure GLUT-5 mRNA expression following 2mM, and 5.5 mM glucose-, and 5.5 mM fructose-treatment for 4 h, and 24 hrespectively. GLUT-5 mRNA was expressed as the fold-change relative toGLUT-5 mRNA levels in 2 mM glucose-treated cells. As depicted in FIG.22, at 4 h, GLUT-5 mRNA levels were low in both the 5.5 mM glucose-, andfructose-treated PANC-1 cells. In contrast, at 24 h, a dramatic increasein GLUT-5 mRNA levels following 5.5 mM fructose-treatment resulted, incomparison to GLUT-5 mRNA levels in 2 mM (1.5 fold higher) or 5.5 mM(1.75 fold higher) glucose-treated cells. This agrees with other studieswhich have reported fructose-induced GLUT-5 mRNA expression in coloncancer (Gouyon F, Onesto C, Dalet V, Pages G, Leturque A, andBrot-Laroche E. Fructose modulates GLUT5 mRNA stability indifferentiated Caco-2 cells: Role of cAMP-signalling pathway and PABP(polyadenylated-binding protein)-interacting protein (Paip) 2. J Biochem375:167-174, 2003). Based on this finding, which requires confirmationin additional pancreatic cancer cell-lines, in addition to demonstrationof GLUT-5 protein expression, the inventors have focused one of in vitrosubaims to characterize the role of the fructose-selective GLUTs(GLUT-2, GLUT-5, and GLUT-7), particularly GLUT-5, in fructose-mediatedpancreatic cancer proliferation.

Example 19 Fructose-Treatment Activates Pancreatic Cancer MAPK Signaling

Several signal transduction pathways may be involved infructose-mediated pancreatic cancer growth but given the importance ofthe MAPK pathway in proliferation, the inventors believed that MAPK wasinvolved. In the MAPK pathway, MEK mediated phosphorylation of thep44/42 MAP kinase (ERK) is the first step in this cascade, which resultsin phosphorylation of p90^(Rsk), leading to transcriptional activationof CREB, and AP1, activating gene transcription and promoting cellulargrowth. The inventors employed Western blot analysis to examine MAPKpathway expression in pancreatic cancer cells incubated in 2 mM glucose,5.5 mM glucose, and 5.5 mM fructose for 48 hours. Levels of the activephosphorylated proteins, pERK, pRSK, and pMEK were normalized to totalERK, RSK, and MEK levels respectively, and compared between thefructose-, and glucose-treated PANC-1 cells. As depicted in FIG. 23,some phosphorylated-ERK, -RSK, and -MEK expression was observed in thePANC-1 cells incubated overnight in 2 mM glucose, and following 48 hincubation in 5.5 mM glucose, an increase in pERK, pRSK, and pMEK levelswas observed. However, highest pERK, pRSK, and pMEK expression wasdemonstrated in PANC-1 cells incubated in 5.5 mM fructose, underpinningthe role of the MAPK pathway in fructose-mediated pancreatic cancer cellproliferation.

In separate experiments PANC-1 cells, and primary cultures of surgicallyresected pancreatic tumors were incubated in 2 mM glucose, or 5.5 mMglucose or 5.5 mM fructose for 48 h, after which nuclear extracts wereincubated with a biotinylated AP-1 probe in binding buffer. AP-1/AP1probe complexes were next conjugated to a streptavidin-coated assayplate and detected using an AP-1 specific antibody, followed by bindingof a horseradish-peroxidase conjugated secondary antibody, and detectionby a colorimetric reagent. As depicted in FIG. 24 a, low levelbackground luciferase activity was demonstrated in vehicle-treated cells(negative control), and high-level luciferase activity was noted in thepositive control, confirming the integrity of the assay.

Following incubation of the PANC-1 cells in 5.5 mM glucose, anapproximate 2-fold increase in luciferase activity was noted incomparison to 2 mM glucose. Highest AP-1 luciferase reporter activitywas observed in the PANC-1 cells that had been incubated in 5.5 mMfructose, which was approximately 40% higher than luciferase levelsmeasured in the PANC-1 cells which had been incubated in 5.5 mM glucose,further demonstrating the importance of MAPK-signalling infructose-mediated pancreatic cancer effects. In a primary culture of apancreatic tumor, prepared as previously described (FIG. 24 b), a2.1-fold increase in AP-1 activation was observed in 5.5 mMfructose-treated cells, versus a 1.8 fold increase in 5.5 mMglucose-treated cells, compared to cells treated with 2 mM glucose.These results provide insight into the mechanism(s) of fructose-inducedpancreatic cancer proliferation, and underscore the role of the MAPKpathway.

Example 20 Pancreatic Cancer Fructose Metabolism Differs Significantlyfrom Glucose

Metabolism comprises anabolic (converting small molecules into big ones)and catabolic processes (converting food into useful energy), predictsthe functional state of a cell, and analyzes the levels of the endproducts (metabolites) of anabolism and catabolism. Metabolic profilingusing stable isotope tracer technology ([1,2-¹³C] sugars) allows themeasurement of the changing pattern of distribution of ¹³C carbons from[1,2-¹³C] glucose in intracellular metabolic intermediates, and providesa simultaneous measure of carbon flow toward the pentose cycle,glycolysis, direct glucose oxidation, tricarboxylic acid (TCA) cycle andfatty acid synthesis (Cascante, M., Boros, L. G., Comin, B., Atauri, P.,Centelles, J. J., Lee, W-N. P. Metabolic control analysis in drugdiscovery and disease. Nature Biotechnology 20: 246-249, 2002; Boros LG, Cascante M, Lee W N. Metabolic profiling of cell growth and death incancer: applications in drug discovery. Drug Discovery Today 7: 364-72,2002). It also reveals specific flux changes in lactate, glutamate,nucleic acid ribose, palmitate and CO₂, and is a useful complement tothe understanding of diseases, including cancer. The inventors employed[1, 2-¹³C₂] glucose, or [1,2-¹³C₂] fructose stable isotope-based dynamicmetabolic profiling (SiDMAP, Los Angeles, US) to track and quantifychanges in glucose, and fructose carbon re-distribution among majormetabolic pathways in pancreatic cancer cells (Lee W N, Boros L G,Puigjaner J, Bassilian S, Lim S, Cascante M. Mass istopomer study oftransketolase-transaldolase pathways of the pentose cycle with[1,2-¹³C₂]glucose. Am J Physiol 274:E843-51, 1998; Lee W N, Byerley L O,Bassilian S, Ajie H O, Clark I, Edmond J. Isotopomer study oflipogenesis in human hepatoma cells in culture: contribution of carbonand hydrogen atoms from glucose. Anal Biochem 226: 100-112). Theprinciples of this methodology are outlined below in a series ofschematics (FIGS. 25-27) illustrating how this technique trackedcarbohydrate metabolism and the data generated. Essentially, there arethree main ultimate metabolic fates for carbohydrates such as glucose orfructose. Firstly, as depicted in FIG. 25, once glucose, and/or fructoseare taken into the cell via the GLUT transporters, they can enterglycolysis (common entry point being fructose 1,6 bisphosphate) togenerate energy (ATP), and lactate.

Secondly, as depicted in FIG. 26, sugars can enter the tricarboxylicacid cycle (TCA) to result in fatty acid synthesis via Acyl-Coa, whichin turn can be metabolized for energy (ATP) generation, and CO₂ release.Thirdly, as depicted in FIG. 27, carbohydrates can be utilized fornucleic acid synthesis via the enzyme transketolase in the pentosephosphate shunt. For the metabolomic studies, 75% confluent cultures ofPANC-1 cells (3×10⁶) were incubated for 24, and 72 h in 2 mM-, or 5.5mM-[1,2-¹³C₂] D-glucose-, or 5.5 mM [1,2-¹³C₂] D-fructose (>99% purity,and 99% isotope enrichment for each carbon position) (Cambridge IsotopeLabs, Massachusetts)-containing media (half unlabeled glucose/fructose,half labeled with the ¹³C tracer) in T75 culture flasks. These timepoints were chosen for the experiments, as previous studies havedemonstrated that carbohydrate metabolism is most active within thistime period. Where possible, these studies were also performed inprimary pancreatic tumor, and normal pancreas cultures. After treatment,culture medium was collected, cells were washed twice in PBS, afterwhich cell pellets were harvested, and mass spectral data were thenobtained on a HP5973 mass selective detector connected to an HP6890 gaschromatograph. An HP-5 capillary column was used for the glucose, riboseand lactate analyses.

Example 21 Pancreatic Cancer Fructose Metabolism Differs Significantlyfrom Glucose

As depicted in FIGS. 28 through 32, the metabolism of fructose andglucose in the pancreatic cancer cells differs significantly. Similarresults were obtained for both the 24 h, and 72 h timepoints, andtherefore, only the 72 h timepoint is depicted. Firstly, thecontribution of glucose and fructose to glycolysis are discussed.Lactate is the main three-carbon product of glycolysis and it is readilysecreted into the cell culture medium, therefore, it can be utilized formeasurement of label incorporation into the three-carbon metabolitepool. Following glucose- or fructose treatment, lactate from thepancreatic cancer cell culture media (0.2 ml) was extracted by ethylenechloride after acidification with HCl, and then derivatized to itspropylamine-heptafluorobutyrate ester form and the m/z 328 (carbons 1-3of lactate) (chemical ionization, CI) were monitored for the detectionof m1 (recycled lactate through the PC) and m2 (lactate produced by theEmbden-Meyerhof-Parnas pathway) for the estimation of pentose cycleactivity (Lee W N, Boros L G, Puigjaner J, Bassilian S, Lim S, CascanteM. Mass istopomer study of transketolase-transaldolase pathways of thepentose cycle with [1, 2-¹³C₂]glucose. Am J Physiol 274:E843-51, 1998).The m1/m2 ratios in lactate produced and released by the PANC-1pancreatic adenocarcinoma cells were recorded in order to determinepentose cycle activity versus anaerobic glycolysis in response toglucose or fructose-treatment. As illustrated in FIG. 28, a largeproportion of the ¹³C-labeled glucose entered glycolysis, and wasmetabolized to generate lactate (FIG. 28 a), and CO₂ (FIG. 28 b). Incontrast, a comparatively small fraction of ¹³C-labeled fructose wasmetabolized to generate lactate and CO₂, resulting in a 3-4 fold lowerlactate and CO₂ production for fructose in comparison to glucose.Secondly, the inventors examined fatty acid synthesis following glucose,and fructose-treatment. The incorporation of ¹³C from [1,2-¹³C] glucoseor fructose gives key information about the fraction of de novolipogenesis in tumor cells and about glucose or fructose carboncontribution to acetyl-CoA for fatty acid synthesis. After glucose- orfructose-treatment of the pancreatic cancer cells, palmitate, stearate,cholesterol and oleate were extracted after saponification of cellpellets in 30% KOH and 100% ethanol using petroleum ether. Fatty acidswere converted to their methylated derivative using 0.5N methanolic-HCl,and palmitate, stearate and oleate were monitored at m/z 270, m/z 298and m/z 264, respectively, with the enrichment of 13C labeled acetylunits to reflect synthesis, elongation and desaturation of the new lipidfraction as determined by mass isotopomer distribution analysis (MIDA)of different isotopomers (Lee W N, Byerley L O, Bassilian S, Ajie H O,Clark I, Edmond J. Isotopomer study of lipogenesis in human hepatomacells in culture: contribution of carbon and hydrogen atoms fromglucose. Anal Biochem 226: 100-112; Lee W N, Lim S, Bassilian S, BergnerE A, Edmond J Fatty acid cycling in human hepatoma cells and the effectsoftrogiitazone. J Biol Chem 273: 20929-20934 1994). As demonstrated inFIG. 29, a significant proportion of glucose metabolism contributed tofatty acid synthesis (FAS) as evidenced by the high levels ofglucose-derived ¹³C-labeled palmitate (FIG. 29 a), stearate (FIG. 29 b),and acetyl CoA (FIG. 29 c), but comparatively less of the ¹³C-labeledfructose was metabolized for FAS generating palmitate (FIG. 29 a),stearate (FIG. 29 b), or acetyl-CoA (FIG. 29 c).

Ribose and deoxyribose synthesis, the building blocks of nucleotides,following glucose, and fructose-treatment were also compared. ¹³Cincorporation from glucose or fructose into RNA ribose or DNAdeoxyribose indicates changes in nucleic acid synthesis rates throughthe respective branches of the pentose cycle. Following the[1,2-¹³C₂]D-glucose-, or [1, 2-13C₂]D-fructose-treatments, RNA ribosewas isolated by acid hydrolysis of cellular RNA after Trizolpurification of cell extracts. Total RNA was quantified byspectrophotometric determination, in triplicate cultures. Ribose wasthen derivatized to its aldonitrile acetate form using hydroxylamine inpyridine with acetic anhydride (Supelco, Bellefonte, Pa.) before massspectral analyses. The ion cluster was monitored around the m/z 256(carbons 1-5 of ribose) (chemical ionization, CI) and m/z 217 (carbons3-5 of ribose) and m/z 242 (carbons 1-4 of ribose) (electron impactionization, EI) to determine molar enrichment and the positionaldistribution of ¹³C in ribose. Ribose molecules labeled with a single¹³C atom on the first carbon position (m1) recovered from RNA were usedto gauge the ribose fraction produced by direct oxidation of glucose orfructose through the G6PD pathway. Ribose molecules labeled with ¹³C onthe first two carbon positions (m2) were used to measure the fractionproduced by transketolase. Doubly labeled ribose molecules (m2 and m4)on the fourth and fifth carbon positions were used to measure molarfraction produced by triose phosphate isomerase and transketolase. Incontrast to the relatively lower contribution of 5.5 mM fructose toglycolysis, and to fatty acid synthesis in comparison to glucose, asdepicted in FIGS. 28, and 29, the metabolomic studies demonstrated that¹³C-labeled 5.5 mM fructose was preferentially metabolized in the PANC-1pancreatic cancer cells via the pentose phosphate shunt to a greaterextent than glucose. As noted above, the pentose phosphate shuntcomprises two pathways; the oxidative pathway, which is regulated byglucose-6-phosphate dehydrogenase (G6PDH), which shuttles 5-carbonsugars back to the glycolytic pathway, and the non-oxidative pathway,which is regulated by the enzyme transketolase (TKK), which drivesnucleic acid synthesis by generation of xylulose-5-phosphate fromfructose-6-phosphate, and glyceraldehyde-3-phosphate. Characterizationof the individual contribution of each sugar to the oxidative (G6PDH),and non-oxidative (TKK) pathways of the pentose phosphate shunt revealedthat whereas 5.5 mM glucose was metabolized primarily via the oxidativecomponent of the pentose phosphate shunt (FIG. 30, first set of bars;glucose), 5.5 mM fructose was preferentially metabolized by TKK (FIG.30, second set of bars; fructose) contributing significantly to nucleicacid synthesis compared to 5.5 mM glucose.

In separate studies, the inventors treated primary cultures of 4pancreatic adenocarcinomas, and 3 normal pancreas tissues with the[1,2-¹³C₂]D-glucose-, or -fructose-tracers as before for 72 h, andcarried out metabolomic studies. A representative experiment is depictedin FIG. 31, and demonstrates that while normal pancreas tissue (leftpanel) effectively oxidizes both 5.5 mM glucose, and fructose togenerate CO₂, the utilization of fructose and glucose for oxidation inthe pancreatic cancer (right panel) is remarkably different, withsignificantly higher glucose oxidation than fructose. In regard to theBRDU-uptake studies in normal pancreas tissues, these metabolomicresults also suggest the differential utilization of fructose versusglucose may be restricted to transformed pancreatic cells.

These fascinating metabolomic results demonstrated for the first timethat metabolism of fructose by pancreatic cancer cells was strikinglydifferent to that of glucose. Although both sugars were utilized to someextent in glycolysis, fatty acid, and nucleic acid synthesis, there aredifferences in the metabolism of glucose, and fructose, at least atthese concentrations, and times, by pancreatic cancer cells. Whereas aconsiderable proportion of administered glucose was metabolized viaglycolysis, and fatty acid synthesis, fructose was in large partmetabolized by the non-oxidative pentose phosphate shunt to synthesizenucleic acids. These results also indicated that the mechanism offructose-mediated increased growth may be more complex and interesting.These results provided important insights into fructose-mediatedincreased pancreatic cancer cell proliferation, and proposed animportant role for the enzyme transketolase.

Example 22 Pancreatic Cancer Cells Utilize Fructose for Purine Synthesis

Based on the metabolomic studies which led the inventors to believe thatfructose was metabolized in pancreatic cancer cells in large part togenerate nucleic acids, the inventors devised a series of experiments toexamine nucleic acid synthesis in the pancreatic cancer cells followingeither glucose or fructose-treatment. PANC-1 cells were incubated asbefore in 5.5 mM glucose or fructose for 24 h, following an ELISA-basedfluorimetric assay (Amplex Red Uric acid uricase assay kit, Invitrogen)was employed to measure uricase activity in conditioned medium derivedfrom the pancreatic cancer cells (Shi Y, Evans J E, Rock K L. Molecularidentification of a danger signal that alerts the immune system to dyingcells Nature 425, 516-21, 2003). Uricase activity correlates highly withuric acid production, a purine by-product of nucleic acid synthesis.Briefly, in the assay, uricase catalyzes the conversion of uric acid toallantoin, hydrogen peroxide (H₂O₂), and carbon dioxide. The H₂O₂ then,in the presence of horseradish peroxidase, reacts stoichiometricallywith Amplex Red reagent to generate the red-fluorescent oxidationproduct, resorufin. Fluoresence was measured in a fluorescencemicroplate reader using excitation at 530 nm and detection at 590 nm,and the assay can detect levels as low as 100 nM uric acid. Asdemonstrated in FIG. 32, uricase activity in conditioned mediumharvested at 96, and 120 h from the fructose-treated PANC-1 cells were˜20%, and ˜50% higher than levels measured in conditioned medium derivedfrom the glucose-treated PANC-1 cells at the same timepoints, p<0.01.These results demonstrated increased pancreatic cancer cell uric acidproduction after fructose-treatment, and as uric acid is a by-product ofpurine metabolism, this observation further supported the notion thatfructose was metabolized to a greater extent than glucose to generatenucleic acids.

Example 23 Fructose-Treatment Increases Pancreatic Cancer TransketolaseExpression and Activity

As noted previously, the metabolomic studies indicated that the enzymetransketolase (TKK) was important in the metabolism of fructose via thepentose phosphate pathway to increase nucleic acid synthesis. Theinventors believe that fructose-treatment of pancreatic cancer cells ledto an increase in TKK-driven nucleic acid synthesis, and thusfructose-treatment would result in an increase in either PANC-1 TKKexpression and/or activity in comparison to equivalent glucoseconcentrations. Western blot analysis on protein lysates derived fromfructose-, and glucose-treated PANC-1 cells were performed (5.5 mMfructose, and glucose for 24, and 48 h as per previous protocol) using aspecific TKK antibody, and compared TKK expression in the fructose-, andglucose-treated PANC-1 cells. As depicted in FIG. 33, TKK expression at24 h, and 48 h in protein lysates derived from 5.5 mM glucose-treatedPANC-1 cells was increased in comparison to expression in 2 mMglucose-treated cells. However, highest TKK expression was observedafter 5.5 mM fructose treatment at 24 h, and 48 h, and TKK levels werealmost 2-fold higher in 5.5 mM fructose-treated, in comparison to 5.5 mMglucose-treated cells (p<0.01). To measure TKK activity, a validatedmethod which calculates TKK enzyme activity from the catalysis of theoxidation of NADH was used (Berrone E, Beltramo E, Solimine C, Ape A U,Porta M. Regulation of intracellular glucose and polyol pathway bythiamine and benfotiamine in vascular cells cultured in high glucose. JBiol. Chem. 7; 281:9307-13. 2006; Comin-Anduix B, Boros L G, Marin S,Boren J, Callol-Massot C, Centelles J J, Torres J L, Agell N, BassilianS, Cascante M. Fermented wheat germ extract inhibits glycolysis/pentosecycle enzymes and induces apoptosis through poly(ADP-ribose) polymeraseactivation in Jurkat T-cell leukemia tumor cells. J Biol. Chem.277:46408-14. 2002; De La Haba G, Leder I G, and Racker E. J. Biol.Chem. 214, 409-426 1995). Briefly, following treatment with 2 mMglucose, 5.5 mM glucose, and 5.5 mM fructose as before, PANC-1 cellswere lysed in Tris-based protein lysis buffer, sonicated, spun, andsupernatants collected. 100 μl of the latter supernatant fractions wereadded to a mixture containing 15 mmol/L ribose 5-phosphate, 50 μmol/LNADH, 0.1 molar Tris-HCL, and 200 units/ml glycerol-3-phosphatedehydrogenase. After gentle mixing the absorbance of the mixture wasmeasured at 10 min intervals for 2 h at 340 nm. Transketolase activitywas then derived from the difference in absorbance at 10, and 80minutes, and expressed as nmol/min/million cells. As depicted in FIG.34, TKK activity in protein lysates derived from 5.5 mM glucose-treatedPANC-1 cells was not increased at 24 h, some increased TKK activity wasobserved following 48, and 72 h 5.5 mM glucose-treatment, but this TKKactivity was not significantly different when compared to the 2 mMglucose-treated cells. In contrast, a striking increase in PANC-1 TKKactivity was demonstrated at all three timepoints (24, 48, and 72 h)following 5.5 mM fructose-treatment, which was 2-fold higher at 24 h,maximally induced (3-fold higher) at 48 h, and 1.7-fold higher at 72 h,in comparison to the activity measured in the 5.5 mM glucose-treatedPANC-1 cells. The transketolase (TKK) enzyme requires nicotinamideadenine dinucleotide (NAD) as a cofactor, a source of which is thiamine.The inventors believe that the addition of oxythiamine, a thiamineinhibitor may abrogate fructose-stimulated pancreatic cancerproliferation if TKK was a key in fructose-mediated pancreatic cancercell growth. Thus, PANC-1 cells were incubated in 2, and 5.5 mM glucose,and 5.5 mM fructose as before for 72 h, with vehicle (H₂O)), and a rangeof oxythiamine concentrations (0.5 to 2 mM). Pancreatic cancerproliferation rates were then measured using the MTS assay (FIG. 35).Fructose-treatment (5.5 mM for 72 h) resulted in increased PANC-1 cellproliferative rates in comparison to those observed following 5.5 mM or2 mM glucose-treatment. Addition of oxythiamine (0.5 mM) inhibitedglucose-induced and fructose-induced PANC-1 proliferation, and supportsthe inventors' belief that the enzyme TKK is important forfructose-induced pancreatic cancer cell proliferation, addition of evenlow oxythiamine concentrations (0.5 mM) virtually abolished thefructose-mediated increase in pancreatic cancer cell proliferation,which also supports the inventors' belief that the enzyme transketolaseis implicated in fructose-mediated increased pancreatic cancer growth.

Example 24 Fructose is Detectable in Human Serum

The normal circulating concentrations of fructose are unknown. To gaininsight as to the potential relevance of the in vitro studies whichdemonstrate a pro-proliferative effect of fructose at concentrationsabove 4-5 mM, the inventors developed a sensitive, and specificELISA-based assay to measure fructose levels in human sera, and tissuefluids. Briefly, aliquots (20 μl) of fructose standards (0.1, 0.5, 1, 2,4, 6, and 8 mM) or unknown serum samples were mixed with 5 μl of a stocksolution (125 U/ml) of the enzyme fructose dehydrogenase (Sigma) in 175μl citric acid (0.05 M) phosphate (0.09 M) buffer (pH 4.5), and 20 μl of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), andphenazine methosulfate (Promega), incubated for 30 minutes, after whichabsorbance at 570 nM was recorded in a 96-well spectrophotometer. Inadditional studies, the inventors confirmed that the assay did notcross-react with glucose, ribose, mannose or xylose, and additionalcontrols included fructose standard samples with added glucose, whichconfirmed that the assay could specifically detect the sugar fructose.As the first analysis, in accordance with institutional review boardguidelines, the inventors obtained 32 serum samples randomly selectedfrom anonymous inpatients at Cedars-Sinai Medical Center. The inventorsalso measured glucose in these same samples using standard glucoseoxidase based methods. No details of medical condition were known forthese patients, and as depicted in FIG. 36, in 24 of these 32 patients,serum fructose was greater than 5 mM. This intriguing result promptedthe inventors to examine serum fructose levels in two other settings.Firstly, the inventors drew fasting blood samples from 6 patients withpancreatic cancer on the morning of surgical tumor resection. Asdepicted in FIG. 37 a, 4 of these 6 patients had fasting serum fructoselevels >5 mM, and two patients had serum fructose concentrations of ˜12mM. The significance of these findings in pancreatic cancer patients isat this point unclear, but the inventors believe that thefructose-mediated pro-proliferative effects seen in pancreatic cancercells in vitro are of significance, given the circulating fructoseconcentrations the inventors measured. In a further experiment, 3 normalvolunteers fasted for 10 hours, an indwelling cannula was placed in aforearm vein, baseline fasting blood drawn, after which they drank twocans of regular soda, which contained 76 g of sugars, almost exclusivelyhigh fructose corn syrup. This source of carbohydrate was chosen toapproximate to equivalent glucose concentrations used in a standard 75 gglucose tolerance test, and to bear relevance that dietary sources ofhigh content refined fructose may pose an independent risk factor forpancreatic cancer growth. As depicted in FIG. 37 b, although meanfasting fructose level was 2.66 mM, within 15 minutes after drinking thetwo sodas, mean serum fructose levels rose to 16.5 mM, peaked at 30minutes at 17 mM, and then fell gradually with a similar excursion toglucose, but serum levels remained higher than 5 mM for the 2 hourduration of blood sampling. Interestingly, although the source of sugarconsumed contained an almost equivalent concentration of glucose, sincehigh fructose corn syrup in 55% fructose, and 45% glucose, serum glucoseconcentrations did not exhibit the same increase across the carbohydratetolerance test. These data may suggest that the capacity tocounter-balance high glucose loads is better than that to counter highfructose loads.

Example 25 Characterize the Mechanism(s) of Fructose-Enhanced PancreaticCancer Proliferation In Vitro

The inventors have performed the experiments in the primary tumorpancreatic cancer PANC-1 cell-line. As the inventors have also observedsimilar fructose-mediated effects on proliferation, MAPK-activation, andsimilar metabolomic profiles in primary cultures of freshly resectedpancreatic tumors, the inventors believe that the PANC-1 cell is a goodmodel to study fructose-mediated effects in pancreatic cancer. Extendingthe studies, and examining proliferative rates and metabolomic profilesin pancreatic cancer cell lines of various phenotypes may be done. Theseinclude MiaPaCa-2, another primary pancreatic cancer cell line, ASPC-1,and HPAF II, pancreatic cancer cell lines derived from ascitic fluid,and Capan1, and Hs766T, both derived from pancreatic cancer metastases.Additionally, the immortalized epithelial cell line derived from normalhuman pancreatic ducts HPDE6, which has previously been shown tomaintain the phenotypic, and genotypic characteristics of normal humanpancreatic ducts (Ouyang H, Mou Lj, Luk C, Liu N, Karaskova J, Squire J,Tsao M S. Immortal human pancreatic duct epithelial cell lines with nearnormal genotype and phenotype. Am J Pathol 157:1623-31, 2000) may beused. For proliferation assays, the pancreatic cells (˜10,000 per well)will be plated onto 96-well plates, in standard 10% FBS, high glucose(25 mM) DMEM medium. Cells are then incubated overnight in 10% FBS, lowglucose (2 mM) DMEM, then switched to medium containing a range ofglucose or fructose concentrations (2.5, 5, 7.5, 10, 15, 25, 50, 75, &100 mM) for times 24, 48, 72 & 96 h. Proliferative rates are measuredusing CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS),according to manufacturers instructions (Promega, Madison, Wis., USA).This rapid assay is ideal for these large numbers of samples and willquickly allow for the determination of optimal dose ranges and treatmenttimes for subsequent experiments. Proliferative rates are alsoquantified by BRDU-uptake in the fructose-, and glucose-treated cells,as employed in the studies. Following the glucose-, andfructose-treatments, aliquots of the pancreatic cancer cells will alsobe prepared for FACS analysis to characterize cell-cycle profiles,determining the percentage of cells in the resting G0/G1 phase, and theproliferative DNA synthesis-(S−) phase. In separate studies, pancreaticcancer cells, primary pancreatic cancers, and normal pancreas cultureswill be treated with the [1,2-¹³C₂]D-glucose-, or -fructose-tracers asbefore for 72 h, and submitted for metabolomic studies.

Example 26 Fructose-Enhanced Pancreatic Cancer Proliferation InvolvesMAPK Activation

Based on the results demonstrating higher pMEK, pRsk, and pCREBexpression, and greater AP-1 luciferase activity in PANC-1 pancreaticcancer cells following fructose-treatment in comparison toglucose-treatment, the inventors believe that the MAPK pathway is a keymediator of fructose-induced pancreatic cancer growth. This hypothesiswill be tested by examining pancreatic cancer MAPK signalling followingglucose, and fructose-treatment in a variety of pancreatic cancer cellsof different phenotypes, and normal pancreatic epithelial cells.Briefly, pancreatic cancer cells are seeded in 6-well plates, andtreated with a range of glucose, and fructose concentrations (2, 5, 7.5,10, 12.5, and 15 mM) for a range of times (6, 12, 24, 48, 72, and 96 h),after which aliquots of protein are harvested in RIPA buffer, andwestern blot analysis employed to measure activation of the MAPKpathway. Image analysis, with densitometric quantitation of immunoblots,is used to measure activation of the MAPK components. For example, theratio of levels of phosphorylated CREB are corrected for total CREBlevels, for each of the treatments, and times and then used to comparethe responsivity of the MAPK pathway to the different sugars in thecell-lines of different phenotypic origins. While not wishing to bebound by any particular theory, the inventors believe that thepancreatic cancer cell-lines with more aggressive phenotypes, such asCapan1, and Hs766T, exhibits greater MAPK activation in response tofructose-treatment, in comparison to the primary pancreatic cancer celllines such as PANC-1. Pharmacological and molecular approaches toregulate MAPK signaling, and examine the effects on proliferation of thepancreatic cancer cells are used. Firstly, the specific MAPK inhibitor,PD 98059 (10-7 to 10-5M), which is added to the pancreatic cancer cellsin combination with the glucose-, and fructose-treatments is used. In asecond approach, zinc-inducible plasmids encoding dominant negative(DN−) MAPK cDNA is employed to downregulate MAPK expression in the celllines which exhibit comparatively high level MAPK expression followingfructose-treatment.

Example 27 Preferential Fructose-Utilization for Nucleic Acid Synthesisis Due to GLUT-5-Mediated Increased Substrate Availability

One question that arises from the metabolomic results is how fructose, a“substrate” leads to increased nucleic acid synthesis, activation ofMAPK signal transduction, and ultimately increased pancreatic cancercell proliferation. While not wishing to be bound by any particulartheory, the inventors believe that the difference in structure offructose (a ketone), and glucose (an aldehyde) plays a role. Analysis ofthe metabolic pathway of fructose reveals that fructose can readily beconverted to fructose-6-phosphate by hexokinase, thereby allowing rapidentry into the pentose phosphate shunt to generate nucleic acids,whereas glucose must first be converted to glucose-6-phosphate, and thenconverted to fructose-6-phosphate by an isomerase. Increasedfructose-mediated nucleic acid synthesis may be a consequence of themolecular structure of fructose, serving as a ketone donor. Thus,efforts to reduce cellular availability of fructose may result inreduced pancreatic cancer proliferative rates as well as theproliferative rates of other cancers. Both glucose and fructose gainentry to the cell via active transport assisted by a family of glucoseuptake and transport proteins (GLUT 1-12). Three of the GLUT proteins(GLUT 2, 5 & 7) show specificity for fructose uptake, and GLUT-5 appearsto be the predominant fructose-transport protein, as reported in severalsolid tumors. No studies have focused on pancreatic cancer. Real-timequantitative RT-PCR is used to measure expression of all twelve GLUTmRNA's (GLUT 1-12) following treatment with a range of glucose, andfructose-concentrations (2, 5, 7.5, 10, 12.5, and 15 mM) for a range oftimes (6, 12, 24, 48, 72, and 96 h). As depicted in the data, FIG. 22,the inventors have already demonstrated that pancreatic cancer GLUT-5mRNA expression is significantly increased following fructose-treatment,and the second hypothesis proposes that altered GLUT-5 expressionfacilitates increased pancreatic cancer fructose-uptake, therebyincreasing availability of fructose substrate for pro-proliferativeactions in the pancreatic cancer cells. Molecular approaches tooverexpress (using Zn-regulatable GLUT-5 expressing plasmids) or silenceGLUT-5 expression (using siRNA approaches) are used and then effects ofthese manipulations on fructose-, and glucose-mediated pancreatic cancercell proliferation, transketolase action (expression and activity), andnucleic acid synthesis (determined by uricase activity, and RNAsynthesis by metabolomic studies using the [1, 2-13C₂]D-glucose-, or-fructose-tracers using the methods employed in the data) are examined.To knock-down GLUT-5 expression, the SureSilencing system (SuperarrayBioscience Corp) which utilizes four distinct interfering RNA's to thetarget is used. These SiRNA's also include an EGFP-tag sequence soclones can be FACS fluorescence sorted. Other silencing systems known inthe art may also be used. Single cell clones (e.g., 30-50) will beexpanded and GLUT-5 expression examined in the pancreatic cancer cellsby western blot, to generate stable pancreatic cancer GLUT-5 knockoutcells. SiRNA approaches to silence other GLUT members that are inducedby fructose, or Zn-inducible vectors to overexpress these otherfructose-selective GLUT's in the pancreatic cancer cells are used.Effects on growth rates observed in the fructose-treated pancreaticcancer cells are also determined. Silenced or increased GLUT expressionare confirmed by Western blot, and the effects of altered GLUTexpression on pancreatic cancer cell growth rates across a range offructose and glucose concentrations are examined. The non-fructoseselective GLUT proteins GLUT-1, and GLUT-3 are employed as negativecontrols in the knock-out, and overexpression experiments.

Example 28 Fructose-Induced Pancreatic Cancer Proliferation RequiresTransketolase

The inventors believe that transketolase is required forfructose-induced cancer proliferation as demonstrated by increasedpancreatic cancer transketolase expression (FIG. 33) and activity (FIG.34) following fructose-treatment, in comparison to glucose-treatment.Pharmacological and molecular approaches to modify transketolaseexpression and to examine effects of altered transketolase activity onfructose-mediated pancreatic cancer proliferative rates are used. Thedata demonstrates that comparatively low (0.5 mM) concentrations of thecompetitive thiamine inhibitor, oxythiamine, almost completely abrogatefructose-induced pancreatic cancer proliferative rates (FIG. 35). Abroader range of oxythiamine concentrations (0.01, 0.005, 0.1, 0.25, and0.5 mM) on fructose-, and glucose-induced pancreatic cancerproliferative rates in the pancreatic cancer cell lines of differentphenotypes are used. SureSilencing system, utilizing 4 distinctinterfering TKK RNA's to block transketolase expression is employed andFACS sorted single cell clones (e.g., 30-50) are expanded, and TKKexpression are examined by western blot. Clones (a minimum of 5 in eachcategory below) with a range of levels of TKK expression are selected(absent TKK, low TKK, moderate TKK, normal TKK levels), treated with 5.5mM glucose, or 5.5 mM fructose as before, and proliferative rates, andmetabolomic profiles are compared between the TKK silenced and wild-typepancreatic cancer cells.

Example 29 Determine Effects of Refined Fructose Consumption on In VivoPancreatic Cancer Growth

The effects of 10% and 20% added refined fructose and glucose onpancreatic cancer development are examined. These percentages are chosenas they are conservative but meaningful equivalent to current humanrefined carbohydrate intake. Therefore, refined glucose or fructose isadministered to mice innoculated subcutaneously with PANC-1 pancreaticcancer cells. Effects of treatments on time to tumor development, andtumor multiplicity are examined.

Example 30 Pancreatic Cancer Xenograft Tumor Model in Nu/Nu Mice

Female athymic Nu/Nu mice are purchased from Jackson Laboratories.PANC-1 cells (3×10⁶) are inoculated subcutaneously on the flank of themice, under isoflurane anesthesia, in accordance with institutionalanimal care and use guidelines. These animals reproducibly develop flankpancreatic tumors between 4-5 weeks. The mice receive a commerciallypurchased standard chow diet (Labdiet.com, certified rodent diet 5002),eat ˜5 g per day, and receive a total daily intake of approximately 1700Kcal, comprising 64.5% CHO, 11.8% fat, 23.5% protein, and 4.6% fiber.Mice also receive a fructose or glucose sugar solution containing 0.42 gor 0.84 g fructose or glucose (diluted in 100 ml) which equate to 10% or20% added refined carbohydrate respectively or vehicle daily from age 3months to 12 months. Developing tumors in the fructose-, glucose-, andvehicle-fed mice are measured twice a week with electronic calipers, andtumor volumes determined by multiplying the square of the width (w) bythe length (l) and dividing by two (i.e., (w²l)/2). Individual animalweights, tumor size and tumor location will be recorded for each animaltwice a week. Animals are euthanized when they develop tumors of 1000mm³ or more or at the end of the experiment, at the same time of day tolimit temporal variations in glucose, insulin, and other parameters. Twohours before killing, the mice are injected intraperitoneally withbromodeoxyuridine (3 mg/mL) in phosphate-buffered saline, (100 μL/10 gbody weight). The end of the experiment is defined as the time when allvehicle-treated mice have developed a tumor. At that time, all remainingmice (vehicle-, glucose-, and fructose-treated) are, tumors resected,and studied.

Example 31 Histology and Biomarker Analysis—Pancreatic Cancer

For histology, pancreatic tumors are fixed in formalin andparaffin-embedded. Tissue sections are processed for immunohistochemicalstaining for the proliferative markers, PCNA, and Ki-67 to compare tumorproliferative rates derived from the fructose-, and glucose-treatedmice. Briefly, tissue sections are deparaffinized in xylene, rehydrated,endogenous peroxidase activity blocked, non-specific binding reducedwith goat serum, after which sections are incubated with antibodies toPCNA or Ki-67 (MIB-1) (1:200). Washed sections are incubated withbiotinylated goat anti-mouse antibody, incubated with the ABC kit(Vector labs), and 3-amino-9-ethylcarbazole to visualize the peroxidasecomplex. Levels of immunostaining are evaluated by visual assessmentwith a semiquantitative scoring system rating staining intensity (from 0to 3). Staining for bromodeoxyuridine (BRDU) is performed with the DAKOAnimal Research Kit system. Briefly, tissue sections are prepared, asabove, and BRDU stained with a mouse anti-BRDU monoclonal antibody(DAKO), slides incubated with streptavidin-horseradish peroxidase, andvisualized with diaminobenzidine chromagen. Counterstaining is performedwith hematoxylin, sections are scored by counting the positive andnegative cells in 10 high-powered fields in tissue samples from 4 micein each group. Results are expressed as the mean percentage with 95% Cl.

Example 32 Glucose, Insulin, IGF1, Lipid, and Inflammatory CytokineLevels

Fructose-, glucose-, and vehicle-treated mice will be fasted for 24 h (9am-9 am) every 4 weeks, and blood will be collected by the tail clipmethod into chilled tubes for assay of glucose, insulin, lipid andinflammatory cytokine levels. Blood will also be collected ateuthanasia. Plasma glucose concentrations will be determined usingGlucotrend 2 (Roche Diagnostics); plasma insulin (Linco Res), and IGF-1Phoenix Pharmaceuticals levels are measured using solid phase two siteenzyme immunoassays; plasma triglyceride, free fatty acids, lactate, andβ-hydroxybutyrate concentrations are analyzed using commerciallyavailable kits (Sigma Diagnostics). Serum inflammatory markers includeC-reactive protein, IL-6 (R&D Systems), serum sialic acid, solubleintracellular and vascular adhesion molecules (sICAM-1, and sVCAM-1)(Bender MedSystems Inc), and amyloid A levels, and are assayed usingcommercially purchased ELISA based kits (R & D, Quantikine). Allparameters are measured in triplicate in the pancreatic cancer cellinnoculated mice, and means compared between the fructose-, andglucose-, and vehicle-fed animals, at the various timepoints using ANOVAwith Bon-Ferroni post t-tests for multiple comparisons. Time curves aregraphed to compare trends in fasting glucose, insulin, lipid, andtriglyceride levels, and inflammatory markers across the dietarytreatment.

While the description above refers to particular embodiments of thepresent invention, it should be readily apparent to people of ordinaryskill in the art that a number of modifications may be made withoutdeparting from the spirit thereof. The accompanying claims are intendedto cover such modifications as would fall within the true spirit andscope of the invention. The presently disclosed embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive, the scope of the invention being indicated by the appendedclaims rather than the foregoing description. All changes that comewithin the meaning of and range of equivalency of the claims areintended to be embraced therein.

1. A method of treating cancer in a mammal, comprising: providing a composition capable of inhibiting and/or regulating a metabolic pathway of fructose; and administering a therapeutically effective amount of the composition to the mammal.
 2. The method of claim 1, wherein the composition is capable of modulating an enzyme in the metabolic pathway of fructose.
 3. The method of claim 1, wherein the composition is capable of modulating hexokinase, fructokinase-1, fructose-1-P adolase, transketolase or an analog thereof.
 4. The method of claim 3, wherein the composition comprises a small interfering RNA (siRNA) capable of suppressing transketolase mRNA expression.
 5. The method of claim 1, wherein the composition comprises a fructose analog adapted to compete with fructose to enter the metabolic pathway of fructose.
 6. The method of claim 1, wherein the composition comprises a thiamine inhibitor.
 7. The method of claim 1, wherein the composition comprises oxythiamine.
 8. A method of treating cancer in a mammal, comprising: providing a composition capable of modulating a GLUT mRNA expression and/or a GLUT function; and administering a therapeutically effective amount of the composition to the mammal.
 9. The method of claim 8, wherein the GLUT is GLUT-5.
 10. The method of claim 8, wherein the composition comprises a small interfering RNA (siRNA) capable of suppressing the GLUT mRNA expression.
 11. The method of claim 8, wherein the composition comprises an antagonist of GLUT.
 12. The method of claim 8, wherein the composition comprises an antagonist of GLUT-5.
 13. A method of treating cancer in a mammal, comprising: providing a fructose-based or a fructose analog-based composition, comprising a fructose or fructose analog conjugated to a compound selected from the group consisting of a toxin, a cell signal deactivator, a radioactive agent and combinations thereof; and administering a therapeutically effective amount of the fructose-based or the fructose-analog based composition to the mammal.
 14. The method of claim 13, wherein the compound is conjugated to fructose at the first or the second carbon atom.
 15. The method of claim 13, wherein the toxin is selected from the group consisting of botulinum, diphtheria and combinations thereof.
 16. The method of claim 13, wherein the deactivator is a cyclin-dependent kinase inhibitor.
 17. The method of claim 13, wherein the radioactive agent is selected from the group consisting of radioactive isotopes of carbon, oxygen, hydrogen, fluorine, iodine, gallium, technetium, indium, copper and combinations thereof.
 18. A method for treating cancer in a mammal, comprising: providing a composition comprising a radioactive isotope of fructose or a fructose analog; and administering a therapeutically effective amount of the composition to the mammal, whereby the radioactive isotope is incorporated into nucleic acid synthesis.
 19. The method of claim 18, wherein the radioactive isotope is selected from the group consisting of carbon-11 (¹¹C), oxygen-15 (¹⁵O) and combinations thereof.
 20. A method for treating cancer in mammal, comprising: providing a fructose analog capable of being cleaved into a toxic metabolite; and administering a therapeutically effective amount of the fructose analog to the mammal, whereby a cellular enzyme converts the fructose analog into the toxic metabolite.
 21. A kit for the treatment of cancer in a mammal, comprising: an agent selected from the group consisting of: a composition capable of inhibiting and/or regulating a metabolic pathway of fructose, a composition capable of modulating a GLUT mRNA expression and/or a GLUT function, a fructose-based or a fructose-analog based composition, wherein the fructose-based or the fructose-analog based composition comprises fructose conjugated to a compound selected from the group consisting of a toxin, a cell signal deactivator, a radioactive agent and combinations thereof, a radioactive isotope of fructose or a fructose analog capable of being incorporated into nucleic acid synthesis, and a fructose analog capable of being cleaved into a toxic metabolite, and combinations thereof; and instructions to use the agent to treat cancer.
 22. The kit of claim 21, wherein the composition capable of inhibiting and/or regulating a metabolic pathway of fructose is a composition capable of modulating hexokinase, fructokinase-1, fructose-1-P adolase, transketolase or an analog thereof.
 23. The kit of claim 21, wherein the composition capable of inhibiting and/or regulating a metabolic pathway of fructose comprises a fructose analog, wherein, upon administration to the mammal, the fructose analog competes with fructose to enter the metabolic pathway of fructose.
 24. The kit of claim 21, wherein the composition capable of modulating the GLUT mRNA expression and/or the GLUT function comprises a small interfering RNA (siRNA) capable of suppressing the GLUT mRNA expression.
 25. The kit of claim 21, wherein the composition capable of modulating the GLUT mRNA expression and/or the GLUT function comprises an antagonist of GLUT. 